Kit comprising an antibody to interleukin 17 receptor-like protein

ABSTRACT

The present invention relates to a novel IL17RLP protein which is a member of the interleukin (IL)-17 receptor family. In particular, isolated nucleic acid molecules are provided encoding the human IL17RLP protein. IL17RLP polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of IL17RLP activity. Also provided are diagnostic methods for detecting immune system-related disorders and therapeutic methods for treating, diagnosing, detecting, and/or preventing immune system-related disorders.

This application is a divisional of U.S. application Ser. No.11/567,524, filed Dec. 6, 2006 (now U.S. Pat. No. 7,638,603, issued Dec.29, 2009), which is a continuation of U.S. application Ser. No.10/645,702, filed Aug. 22, 2003 now abandoned, which is a divisional ofU.S. application Ser. No. 09/796,844, filed Mar. 2, 2001 (now U.S. Pat.No. 6,849,719, issued Feb. 1, 2005), which claims benefit under 35U.S.C. §119(e) of U.S. Provisional Application No. 60/187,015, filedMar. 6, 2000; U.S. application Ser. No. 09/796,844 is also acontinuation-in-part of International Application No. PCT/US00/05759,filed Mar. 6, 2000 (now abandoned), and a continuation-in-part ofInternational Application No. PCT/US99/21048, filed Sep. 15, 1999, and acontinuation-in-part of U.S. application Ser. No. 09/268,311, filed Mar.16, 1999 (now U.S. Pat. No. 6,482,923, issued Nov. 19, 2002);International Application No. PCT/US00/05759 is a continuation-in-partof U.S. application Ser. No. 09/268,311, filed Mar. 16, 1999 (now U.S.Pat. No. 6,482,923, issued Nov. 19, 2002); International Application No.PCT/US99/21048 is a continuation-in-part of U.S. application Ser. No.09/268,311 (now U.S. Pat. No. 6,482,923, issued Nov. 19, 2002). Each ofthese applications is hereby incorporated by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted substitute sequence listing(Name: SubstituteSequenceListing.txt, Size: 37,686 kilobytes; and Dateof Creation: Dec. 30, 2010) is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to a novel human gene encoding apolypeptide which is a member of the interleukin (IL)-17 receptorfamily. More specifically, isolated nucleic acid molecules are providedencoding a human polypeptide named Interleukin 17-Receptor-Like Protein,hereinafter referred to as IL17RLP. IL17RLP polypeptides are alsoprovided, as are vectors, host cells and recombinant methods forproducing the same. Also provided are diagnostic methods for diagnosingand/or detecting disorders related to the immune system and therapeuticmethods for treating and/or preventing such disorders. The inventionfurther relates to screening methods for identifying agonists andantagonists of IL17RLP activity.

BACKGROUND OF THE INVENTION

Cytokines typically exert their respective biochemical and physiologicaleffects by binding to specific receptor molecules. Receptor binding willthen stimulate specific signal transduction pathways (Kishimoto, T., etal., Cell 76:253-262 (1994). The specific interactions of cytokines withtheir receptors are often the primary regulators of a wide variety ofcellular process including activation, proliferation, anddifferentiation (Arai, K.-I, et al., Ann. Rev. Biochem. 59:783-836(1990); Paul, W. and Seder, R., Cell 76:241-251 (1994)).

Human interleukin (IL)-17 was only recently identified. IL-17 is a 155amino acid polypeptide which was molecularly cloned from a CD4+ T-cellcDNA library (Yao, Z., et al., J. Immunol. 155:5483-5486 (1995)). TheIL-17 polypeptide contains an N-terminal signal peptide and containsapproximately 72% identity at the amino acid level with a T-cell trophicherpesvirus saimiri (HVS) gene designated HVS13. High levels of IL-17are secreted from CD4-positive primary peripheral blood leukocytes (PBL)upon stimulation (Yao, Z., et al., Immunity 3:811-821 (1995)). Treatmentof fibroblasts with IL-17, HVS13, or another murine homologue,designated CTLA8, activate signal transduction pathways and result inthe stimulation of the NF-kappaB transcription factor family, thesecretion of IL-6, and the costimulation of T-cell proliferation (Yao,Z., et al., Immunity 3:811-821 (1995)).

An HVS13-Fc fusion protein was used to isolate a murine IL-17 receptormolecule which does not appear to belong to any of the previouslydescribed cytokine receptor families (Yao, Z., et al., Immunity3:811-821 (1995)). The murine IL-17 receptor (mIL-17R) is predicted toencode a type I transmembrane protein of 864 amino acids with anapparent molecular mass of 97.8 kDa. mIL-17R is predicted to possess anN-terminal signal peptide with a cleavage site between alanine-31 andserine-32. The molecule also contains a 291 amino acid extracellulardomain, a 21 amino acid transmembrane domain, and a 521 amino acidcytoplasmic tail. A soluble recombinant IL-17R molecule consisting of323 amino acids of the extracellular domain of IL-17R fused to the Fcportion of human IgG1 was able to significantly inhibit IL-17-inducedIL-6 production by murine NIH-3T3 cells (supra).

Interestingly, the expression of the IL-17 gene is highly restricted. Itis typically observed primarily in activated T-lymphocyte memory cells(Broxmeyer, H. J. Exp. Med. 183:2411-2415 (1996); Fossiez, F., et al.,J. Exp. Med. 183:2593-2603 (1996)). Conversely, the IL-17 receptorappears to be expressed in a large number of cells and tissues including(Rouvier, E., et al., J. Immunol. 150:5445-5456 (1993); Yao, Z., et al.,J. Immunol. 155:5483-5486 (1995)). It remains to be seen, however, ifIL-17 itself can play an autocrine role in the expression of IL-17.IL-17 has been implicated as a causitive agent in the expression ofIL-6, IL-8, G-CSF, Prostaglandin E (PGE₂), and intracellular adhesionmolecule (ICAM)-1 (Fossiez, F., supra; Yao, Z., et al., Immunity3:811-821 (1995)). Each of these molecules possesses highly relevant andpotentially therapeutically valuable properties. For instance, IL-6 isinvolved in the regulation of hematopoietic stem and progenitor cellgrowth and expansion (Ikebuchi, K., et al., Proc. Natl. Acad. Sci. USA84:9035-9039 (1987); Gentile, P. and Broxmeyer, H. E. Ann. N.Y. Acad.Sci. USA 628:74-83 (1991)). IL-8 exhibits a myelosuppressive activityfor stem and immature subsets of myeloid progenitors (Broxmeyer, H. E.,et al., Ann. Hematol. 71:235-246 (1995); Daly, T. J., et al., J. Biol.Chem. 270:23282-23292 (1995)). G-CSF acts early and late to activate andstimulate hematopoiesis in general (more specifically, neutrophilhematopoiesis) while PGE₂ enhances erythropoiesis, suppresseslymphopoiesis and myelopoiesis in general, and strongly suppressesmonocytopoiesis (Broxmeyer, H. E. Amer. J. Ped. Hematol./Oncol. 14:22-30(1992); Broxmeyer, H. E. and Williams, D. E. CRC Crit. Rev.Oncol./Hematol. 8:173-226 (1988)).

IL-17 receptor appears to be structurally unrelated to any previouslydescribed cytokine receptor family. Despite the existence of 12 cysteineresidues in the extracellular domain, their relative positions are notcharacteristic of receptor molecules classified as members of theimmunoglobulin superfamily (Williams, A. and Barclay, A. Annu. Rev.Immunol. 6:381-405 (1988)), the TNFR family (Smith, C., et al., Science248:1019-1023 (1990)), the hematopoietin receptor family (Cosman, D.Cytokine 5:95-106 (1993)), or any previously described tyrosine kinasereceptors (Hanks, S., et al., Science 241:42-52 (1988)).

Thus, there is a need for polypeptides that function as receptormolecules for cytokines and, thereby, function in the transfer of anextracellular signal ultimately to the nucleus of the cell, sincedisturbances of such regulation may be involved in disorders relating tocellular activation, hemostasis, angiogenesis, tumor metastasis,cellular migration and ovulation, as well as neurogenesis. Therefore,there is a need for identification and characterization of such humanpolypeptides which can play a role in detecting, preventing,ameliorating or correcting such disorders.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding at least a portion of the IL17RLPpolypeptide having the complete amino acid sequence shown in SEQ ID NO:2or the complete amino acid sequence encoded by the cDNA clone depositedas plasmid DNA with the American Type Culture Collection (ATCC™) asATCC™ Deposit Number 209198 on Aug. 8, 1997. The ATCC™ is located at10801 University Boulevard, Manassas, Va. 20110-2209, USA. Thenucleotide sequence determined by sequencing the deposited IL17RLPclone, which is shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1), contains anopen reading frame encoding a complete polypeptide of 426 amino acidresidues, including an initiation codon encoding an N-terminalmethionine at nucleotide positions 10-12, and a predicted molecularweight of about 47.1 kDa. Nucleic acid molecules of the inventioninclude those encoding the complete amino acid sequence excepting theN-terminal methionine shown in SEQ ID NO:2, or the complete amino acidsequence excepting the N-terminal methionine encoded by the cDNA clonein ATCC™ Deposit Number 209198, which molecules also can encodeadditional amino acids fused to the N-terminus of the IL17RLP amino acidsequence.

The encoded polypeptide has a predicted leader sequence of 19 aminoacids underlined in FIGS. 1A, 1B, and 1C; and the amino acid sequence ofthe predicted mature IL17RLP protein is also shown in FIGS. 1A, 1B, and1C as amino acid residues 20-426, and as residues IL-407 in SEQ ID NO:2.

In another embodiment, the encoded polypeptide has a predicted leadersequence from Met-(−19) to Ser-(−6) of SEQ ID NO:2 (i.e., from Met-1 toSer-14 of the amino acid sequence presented in FIGS. 1A, 1B, and 1C); anextracellular domain from Ala-(−5) to Trp-271 of SEQ ID NO:2 (i.e., fromAla-15 to Tyr-290 of the amino acid sequence presented in FIGS. 1A, 1B,and 1C); a transmembrane domain from Leu-272 to Leu-292 of SEQ ID NO:2(i.e., from Leu-291 to Leu-311 of the amino acid sequence presented inFIGS. 1A, 1B, and 1C); and an intracellular domain from Met-293 toLeu-407 of SEQ ID NO:2 (i.e., from Met-312 to Leu-426 of the amino acidsequence presented in FIGS. 1A, 1B, and 1C). The predicted length of theleader peptide in this embodiment is within the originally predictedrange of 14-19 amino acids.

In an additional embodiment, the IL17RLP transmembrane domain may havean N-terminal boundary beginning at amino acid residue Pro-268, Gly-269,Gly-270, Trp-271 or Leu-272 of the IL17RLP sequence as shown in SEQ IDNO:2 (i.e., amino acid residues Pro-287, Gly-288, Gly-289, Trp-290 orLeu-291 of the IL17RLP sequence as shown in FIGS. 1A, 1B, and 1C) and aC-terminal boundary including amino acid residue Tyr-291, Leu-292,Met-293 or Trp-294 of the IL17RLP sequence as shown in SEQ ID NO:2(i.e., amino acid residues Tyr-310, Leu-311, Met-312 or Trp-313 of theIL17RLP sequence as shown in FIGS. 1A, 1B, and 1C).

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising, or alternatively consisting of, a polynucleotidecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence encoding the IL17RLP polypeptide having thecomplete amino acid sequence in SEQ ID NO:2 (i.e., positions −19 to 407of SEQ ID NO:2); (b) a nucleotide sequence encoding the IL17RLPpolypeptide having the complete amino acid sequence in SEQ ID NO:2excepting the N-terminal methionine (i.e., positions −18 to 407 of SEQID NO:2); (c) a nucleotide sequence encoding the predicted matureIL17RLP polypeptide having the amino acid sequence at positions 1 to 407in SEQ ID NO:2; (d) a nucleotide sequence encoding a polypeptidecomprising the predicted extracellular domain of the IL17RLP polypeptidehaving the amino acid sequence at positions 1 to 271 in SEQ ID NO:2; (e)a nucleotide sequence encoding a soluble IL17RLP polypeptide having thepredicted extracellular and intracellular domains, but lacking thepredicted transmembrane domain; (f) a nucleotide sequence encoding theIL17RLP polypeptide having the complete amino acid sequence encoded bythe cDNA clone contained in ATCC™ Deposit No. 209198; (g) a nucleotidesequence encoding the IL17RLP polypeptide having the complete amino acidsequence excepting the N-terminal methionine encoded by the cDNA clonecontained in ATCC™ Deposit No. 209198; (h) a nucleotide sequenceencoding the mature IL17RLP polypeptide having the amino acid sequenceencoded by the cDNA clone contained in ATCC™ Deposit No. 209198; (i) anucleotide sequence encoding the extracellular domain of the IL17RLPpolypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC™ Deposit No. 209198; and (j) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), (h) or (i) above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise, or alternatively consist of, a polynucleotidehaving a nucleotide sequence at least 80%, 85%, or 90% identical, andmore preferably at least 95%, 96%, 97%, 98%, 99% or 100% identical, toany of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g),(h) or (i), above, or a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide in (a), (b), (c), (d), (e),(f), (g), (h) or (i), above. This polynucleotide which hybridizes doesnot hybridize under stringent hybridization conditions to apolynucleotide having a nucleotide sequence consisting of only Aresidues or of only T residues. An additional nucleic acid embodiment ofthe invention relates to an isolated nucleic acid molecule comprising apolynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a IL17RLP polypeptide having an amino acidsequence in (a), (b), (c), (d), (e), (f), (g) or (h), above.

An additional nucleic acid embodiment of the invention relates to anisolated nucleic acid molecule comprising, or alternatively consistingof, a polynucleotide which encodes the amino acid sequence of anepitope-bearing portion of a IL17RLP polypeptide having an amino acidsequence in (a), (b), (c), (d), (e), (f) or (g), above. A furtherembodiment of the invention relates to an isolated nucleic acid moleculecomprising a polynucleotide which encodes the amino acid sequence of aIL17RLP polypeptide having an amino acid sequence which contains atleast one amino acid substitution, but not more than 50 amino acidsubstitutions, even more preferably, not more than 40 amino acidsubstitutions, still more preferably, not more than 30 amino acidsubstitutions, and still even more preferably, not more than 20 aminoacid substitutions. Of course, in order of ever-increasing preference,it is highly preferable for a polynucleotide which encodes the aminoacid sequence of a IL17RLP polypeptide to have an amino acid sequencewhich contains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acidsubstitutions. Conservative substitutions are preferable.

In another embodiment, the present invention includes a polynucleotideof 1,918 nucleotides (SEQ ID NO:17) which encodes the IL17RLPpolypeptide provided in SEQ ID NO:18. The IL17RLP of SEQ ID NO:18differs from the IL17RLP provided in SEQ ID NO:2 only by the deletion ofthe C-terminal two residues (Cys-406 and Leu-407 of SEQ ID NO:2) and theaddition of nine amino acid residues (Leu-425 through Ile-433 of SEQ IDNO:18). The extracellular domain of IL17RLP is identical in SEQ ID NO:2and SEQ ID NO:18. The IL17RLP polynucleotide sequence shown in SEQ IDNO:17 was derived from sequencing the HAPOR40 cDNA clone deposited withthe ATCC™ with ATCC™ Deposit No. 209198 on Aug. 8, 1997.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofIL17RLP polypeptides or peptides by recombinant techniques.

In accordance with a further aspect of the present invention, there isprovided a process for producing such polypeptide by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing an IL17RLP nucleic acid sequence,under conditions promoting expression of said protein and subsequentrecovery of said protein.

The invention further provides an isolated IL17RLP polypeptidecomprising, or alternatively consisting of, an amino acid sequenceselected from the group consisting of: (a) the amino acid sequence ofthe full-length IL17RLP polypeptide having the complete amino acidsequence shown in SEQ ID NO:2 (i.e., positions −19 to 407 of SEQ IDNO:2); (b) the amino acid sequence of the full-length IL17RLPpolypeptide having the complete amino acid sequence shown in SEQ ID NO:2excepting the N-terminal methionine (i.e., positions −18 to 407 of SEQID NO:2); (c) the amino acid sequence of the mature IL17RLP polypeptidehaving the complete amino acid sequence shown in SEQ ID NO:2 (i.e.,positions 1 to 407 of SEQ ID NO:2); (d) the amino acid sequence of thepredicted extracellular domain of the IL17RLP polypeptide having thecomplete amino acid sequence shown in SEQ ID NO:2 (i.e., positions 1 to271 of SEQ ID NO:2); (e) the amino acid sequence of a soluble IL17RLPpolypeptide having the predicted extracellular and intracellulardomains, but lacking the predicted transmembrane domain; (f) thecomplete amino acid sequence encoded by the cDNA clone contained in theATCC™ Deposit No. 209198; (g) the complete amino acid sequence exceptingthe N-terminal methionine encoded by the cDNA clone contained in theATCC™ Deposit No. 209198; (h) the complete amino acid sequence of themature IL17RLP encoded by the cDNA clone contained in the ATCC™ DepositNo. 209198, and; (i) the complete amino acid sequence of theextracellular domain of the IL17RLP encoded by the cDNA clone containedin the ATCC™ Deposit No. 209198. The polypeptides of the presentinvention also include polypeptides having an amino acid sequence atleast 80% or 85% identical, more preferably at least 90% identical, andstill more preferably 95%, 96%, 97%, 98%, 99% or 100% identical to thosedescribed in (a), (b), (c), (d), (e), (f), (g), (h) or (i) above, aswell as polypeptides having an amino acid sequence with at least 90%similarity, and more preferably at least 95% similarity, to those above.

An additional embodiment of this aspect of the invention relates to apeptide or polypeptide which comprises, or alternatively consists of,the amino acid sequence of an epitope-bearing portion of a IL17RLPpolypeptide having an amino acid sequence described in (a), (b), (c),(d), (e), (f), (g), (h) or (i), above. Peptides or polypeptides havingthe amino acid sequence of an epitope-bearing portion of an IL17RLPpolypeptide of the invention include portions of such polypeptides withat least six or seven, preferably at least nine, and more preferably atleast about 30 amino acids to about 50 amino acids, althoughepitope-bearing polypeptides of any length up to and including theentire amino acid sequence of a polypeptide of the invention describedabove also are included in the invention.

A further embodiment of the invention relates to a polypeptide whichcomprises, or alternatively consists of, the amino acid sequence of anIL17RLP polypeptide having an amino acid sequence which contains atleast one amino acid substitution, but not more than 50 amino acidsubstitutions, even more preferably, not more than 40 amino acidsubstitutions, still more preferably, not more than 30 amino acidsubstitutions, and still even more preferably, not more than 20 aminoacid substitutions. Of course, in order of ever-increasing preference,it is highly preferable for a peptide or polypeptide to have an aminoacid sequence which comprises the amino acid sequence of an IL17RLPpolypeptide, which contains at least one, but not more than 20, 10, 9,8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In specificembodiments, the number of additions, substitutions, and/or deletions inthe amino acid sequence of FIGS. 1A, 1B, and 1C, or fragments thereof(e.g., the mature form and/or other fragments described herein), is 1-5,5-10, 5-25, 5-50, 10-50, 50-150, 50-200 or 100-250, conservative aminoacid substitutions are preferable.

In another embodiment, the invention provides an isolated antibody thatbinds specifically to a IL17RLP polypeptide having an amino acidsequence described in (a), (b), (c), (d), (e), (f), (g), (h) or (i)above. The invention further provides methods for isolating antibodiesthat bind specifically to a IL17RLP polypeptide having an amino acidsequence as described herein. Such antibodies are useful diagnosticallyor therapeutically as described below.

The invention also provides for pharmaceutical compositions comprisingIL17RLP polynucleotides or polypeptides, particularly human IL17RLPpolynucleotides or polypeptides, which may be employed, for instance, totreat, diagnose, detect, and/or prevent disorders relating to cellularactivation, hemostasis, angiogenesis, tumor metastasis, cellularmigration and ovulation, as well as neurogenesis. Methods of treating,diagnosing, and/or detecting individuals in need of IL17RLPpolynucleotides or polypeptides are also provided.

The invention further provides compositions comprising an IL17RLPpolynucleotide or an IL17RLP polypeptide for administration to cells invitro, to cells ex vivo and to cells in vivo, or to a multicellularorganism. In certain particularly preferred embodiments of this aspectof the invention, the compositions comprise an IL17RLP polynucleotidefor expression of an IL17RLP polypeptide in a host organism fortreatment, diagnosis, detection, and/or prevention of disease.Particularly preferred in this regard is expression in a human patientfor treatment, diagnosis, detection, and/or prevention of a dysfunctionassociated with aberrant endogenous activity of an IL17RLP polypeptide.

The present invention also provides a screening method for identifyingcompounds capable of enhancing or inhibiting a biological activity ofthe IL17RLP polypeptide, which involves contacting a ligand which isinhibited by the IL17RLP polypeptide with the candidate compound in thepresence of an IL17RLP polypeptide, assaying receptor-binding activityof the ligand in the presence of the candidate compound and of IL17RLPpolypeptide, and comparing the ligand activity to a standard level ofactivity, the standard being assayed when contact is made between theligand itself in the presence of the IL17RLP polypeptide and the absenceof the candidate compound In this assay, an increase in ligand activityover the standard indicates that the candidate compound is an agonist ofIL17RLP activity and a decrease in ligand activity compared to thestandard indicates that the compound is an antagonist of IL17RLPactivity.

In another aspect, a screening assay for agonists and antagonists isprovided which involves determining the effect a candidate compound hason IL17RLP binding to a ligand. In particular, the method involvescontacting the ligand with an IL17RLP polypeptide and a candidatecompound and determining whether IL17RLP polypeptide binding to theligand is increased or decreased due to the presence of the candidatecompound. In this assay, an increase in binding of IL17RLP over thestandard binding indicates that the candidate compound is an agonist ofIL17RLP binding activity and a decrease in IL17RLP binding compared tothe standard indicates that the compound is an antagonist of IL17RLPbinding activity.

It has been discovered that IL17RLP is expressed not only in adultpulmonary tissue, but also in Crohn's Disease tissue, kidney pyramid,cortex, and medulla tissues, hippocampus, frontal cortex of the brainfrom a patient with epilepsy, adrenal gland tumor, striatum depression,osteclastoma, endometrial tumor, and hypothalamus from a patient withSchizophrenia. Therefore, nucleic acids of the invention are useful ashybridization probes for differential identification of the tissue(s) orcell type(s) present in a biological sample. Similarly, polypeptides andantibodies directed to those polypeptides are useful to provideimmunological probes for differential identification of the tissue(s) orcell type(s). In addition, for a number of disorders of the abovetissues or cells, particularly of the immune system, significantlyhigher or lower levels of IL17RLP gene expression may be detected incertain tissues (e.g., cancerous and wounded tissues) or bodily fluids(e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken froman individual having such a disorder, relative to a “standard” IL17RLPgene expression level, i.e., the IL17RLP expression level in healthytissue from an individual not having the immune system disorder. Thus,the invention provides a diagnostic method useful during diagnosis ofsuch a disorder, which involves: (a) assaying IL17RLP gene expressionlevel in cells or body fluid of an individual; (b) comparing the IL17RLPgene expression level with a standard IL17RLP gene expression level,whereby an increase or decrease in the assayed IL17RLP gene expressionlevel compared to the standard expression level is indicative ofdisorder in the immune system.

An additional aspect of the invention is related to a method fortreating, diagnosing, and/or detecting an individual in need of anincreased level of IL17RLP activity in the body comprising administeringto such an individual a composition comprising a therapeuticallyeffective amount of an isolated IL17RLP polypeptide of the invention oran agonist thereof.

A still further aspect of the invention is related to a method fortreating, diagnosing, and/or detecting an individual in need of adecreased level of IL17RLP activity in the body comprising,administering to such an individual a composition comprising atherapeutically effective amount of an IL17RLP antagonist. Preferredantagonists for use in the present invention are IL17RLP-specificantibodies.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, and 1C show the nucleotide sequence (SEQ ID NO:1) anddeduced amino acid sequence (SEQ ID NO:2) of IL17RLP.

The predicted leader sequence of about 19 amino acids is underlined.Note that the methionine residue at the beginning of the leader sequencein FIGS. 1A, 1B, and 1C is shown in position number (positive) 1,whereas the leader positions in the corresponding sequence of SEQ IDNO:2 are designated with negative position numbers. Thus, the leadersequence positions 1 to 19 in FIGS. 1A, 1B, and 1C correspond topositions −19 to −1 in SEQ ID NO:2.

Six potential asparagine-linked glycosylation sites are marked in theamino acid sequence of IL17RLP. The sites are marked with the bold poundsymbol (#) above the nucleotide sequence coupled with a bolded oneletter abbreviation for the asparagine (N) in the amino acid sequence inFIGS. 1A, 1B, and 1C; that is, the actual asparagine residues which arepotentially glycosylated is bolded in FIGS. 1A, 1B, and 1C. Thepotential N-linked glycosylation sequences are found at the followinglocations in the IL17RLP amino acid sequence: N-67 through W-70 (N-67,V-68, S-69, W-70); N-103 through E-106 (N-103, Y-104, T-105, E-106;N-156 through S-159 (N-156, F-157, T-158, S-159); N-183 through A-186(N-183, I-184, T-185, A-186); N-197 through T-200 (N-197, F-198, T-199,T-200); and N-283 through K-286 (N-283, K-284, S-285, K-286). Twopotential cAMP- and cGMP-dependent protein kinase phosphorylation sitesare also marked in FIGS. 1A, 1B, and 1C with a bolded lysine symbol (K)in the IL17RLP amino acid sequence and an asterisk (*) above the firstnucleotide encoding that lysine residue in the IL17RLP nucleotidesequence. The potential cAMP- and cGMP-dependent protein kinasephosphorylation sequences are found in the IL17RLP amino acid sequenceat the following locations: K-141 through threonine-231 (K-228, K-229,Q-230, T-231) and K-319 through S-322 (K-319, K-320, T-321, S-322).Three potential Protein Kinase C (PKC) phosphorylation sites are alsomarked in FIGS. 1A, 1B, and 1C with a bolded serine or tyrosine symbol(S or T) in the IL17RLP amino acid sequence and an asterisk (*) abovethe first nucleotide encoding that serine tyrosine residue in theIL17RLP nucleotide sequence. The potential PKC phosphorylation sequencesare found in the IL17RLP amino acid sequence at the following locations:S-77 through R-79 (S-77, 1-78, R-79); T-89 through K-91 (T-89, G-90,K-91); and T-384 through K-386 (T-384, Q-385, K-386). Three potentialCasein Kinase II (CK2) phosphorylation sites are also marked in FIGS.1A, 1B, and 1C with a bolded serine symbol (S) in the IL17RLP amino acidsequence and an asterisk (*) above the first nucleotide encoding theappropriate serine residue in the IL17RLP nucleotide sequence. Thepotential CK2 phosphorylation sequences are found at the followinglocations in the IL17RLP amino acid sequence: S-178 through D-181(S-178, L-179, W-180, D-181); S-402 through D-405 (S-402, V-403, C-404,D-405); and S-414 through E-417 (S-414, P-415, S-416, E-417). A singlepotential myristylation site is found in the IL17RLP amino acid sequenceshown in FIGS. 1A, 1B, and 1C. The potential myristylation site ismarked in FIGS. 1A, 1B, and 1C with a double underline delineating theamino acid residues representing the potential myristolation site in theIL17RLP amino acid sequence. The potential myristolation site is locatedat the following position in the IL17RLP amino acid sequence: G-116through F-121 (G-116, G-117, K-118, W-119, T-120, F-121).

Mutations in one or more of the amino acid residues in the above-recitedpotential structural features of the IL17RLP polypeptide arecontemplated as mutations which may affect biological, structural,binding or other characteristics of an IL17RLP DNA or polypeptide of theinvention.

FIG. 2 shows the regions of identity between the amino acid sequences ofthe IL17RLP protein and translation product of the murine mRNA for IL-17receptor (SEQ ID NO:3), determined by the computer program Bestfit(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711) using the default parameters.

FIG. 3 shows an analysis of the IL17RLP amino acid sequence. Alpha,beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index or Jameson-Wolf” graph,the positive peaks indicate locations of the highly antigenic regions ofthe IL17RLP protein, i.e., regions from which epitope-bearing peptidesof the invention can be obtained.

In the “Antigenic Index or Jameson-Wolf” graph, the positive peaksindicate locations of the highly antigenic regions of the IL17RLPprotein, i.e., regions from which epitope-bearing peptides of theinvention can be obtained. Non-limiting examples of antigenicpolypeptides or peptides that can be used to generate IL17RLP-specificantibodies include: a polypeptide comprising amino acid residues fromabout a polypeptide comprising amino acid residues from about Ser-14 toabout Val-22 in SEQ ID NO:2, a polypeptide comprising amino acidresidues from about Cys-24 to about Pro-32 in SEQ ID NO:2, a polypeptidecomprising amino acid residues from about Ile-41 to about Arg-49 in SEQID NO:2, a polypeptide comprising amino acid residues from about Thr-89to about Val-97 in SEQ ID NO:2, a polypeptide comprising amino acidresidues from about Thr-110 to about Lys-118 in SEQ ID NO:2, apolypeptide comprising amino acid residues from about Ala-144 to aboutSer-152 in SEQ ID NO:2, a polypeptide comprising amino acid residuesfrom about Thr-240 to about Val-248 in SEQ ID NO:2, a polypeptidecomprising amino acid residues from about Gly-258 to about Thr-267 inSEQ ID NO:2, a polypeptide comprising amino acid residues from aboutLeu-280 to about Gly-288 in SEQ ID NO:2, a polypeptide comprising aminoacid residues from about Cys-404 to about Glu-412 in SEQ ID NO:2, apolypeptide comprising amino acid residues from about Pro-415 to aboutSer-423 in SEQ ID NO:2, a polypeptide comprising amino acid residuesfrom about Gly-409 to about Glu-417 in SEQ ID NO:2, and a polypeptidecomprising amino acid residues from about Cys-404 to about Leu-426 inFIGS. 1A, 1B, and 1C (which is identical to the sequence shown in SEQ IDNO:2 with exception to the numbering schemes as detailed above).

The data presented in FIG. 3 are also represented in tabular form inTable I. The data presented in Table I is identical to that originallypresented in FIG. 3. The columns are labeled with the headings “Res”,“Position”, and Roman Numerals I-XIV. The column headings refer to thefollowing features of the amino acid sequence presented in FIG. 3 andTable I: “Res”: amino acid residue of SEQ ID NO:2 or FIGS. 1A, 1B, and1C (which is the identical sequence shown in SEQ ID NO:2, with theexception that the residues are numbered 1-426 in FIGS. 1A, 1B, and 1Cand −19 through 407 in SEQ ID NO:2); “Position”: position of thecorresponding residue within SEQ ID NO:2 or FIGS. 1A, 1B, and 1C (whichis the identical sequence shown in SEQ ID NO:2, with the exception thatthe residues are numbered 1-366 in FIGS. 1A, 1B, and 1C and −19 through407 in SEQ ID NO:2); I: Alpha, Regions—Garnier-Robson; II: Alpha,Regions—Chou-Fasman; III: Beta, Regions—Garnier-Robson; IV: Beta,Regions—Chou-Fasman; V: Turn, Regions—Garnier-Robson; VI: Turn,Regions—Chou-Fasman; VII: Coil, Regions—Garnier-Robson; VIII:Hydrophilicity Plot—Kyte-Doolittle; IX: Hydrophobicity Plot—Hopp-Woods;X: Alpha, Amphipathic Regions—Eisenberg; XI: Beta, AmphipathicRegions—Eisenberg; XII: Flexible Regions—Karplus-Schulz; XIII: AntigenicIndex—Jameson-Wolf; and XIV: Surface Probability Plot—Emini.

DETAILED DESCRIPTION

The present invention provides isolated nucleic acid moleculescomprising, or alternatively consisting of, a polynucleotide encoding aIL17RLP polypeptide having the amino acid sequence shown in SEQ ID NO:2,which was determined by sequencing a cloned cDNA. The nucleotidesequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) was obtained bysequencing the HAPOR40 clone, which was deposited on Aug. 8, 1997 at theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209, and given accession number ATCC™ 209198. The depositedclone is contained in the pBLUESCRIPT™ SK(−) plasmid (STRATAGENE™, LaJolla, Calif.).

The IL17RLP protein of the present invention shares sequence homologywith the translation product of the murine mRNA for IL-17 receptor (FIG.2; SEQ ID NO:3). Murine IL-17 receptor is thought to be an importantcomponent of the IL-17 cytokine signal transduction pathway. IL-17receptor appears to be structurally unrelated to any members ofpreviously described cytokine receptor families. The IL-17/IL-17receptor complex activates NF-kappaB activity. NF-kappaB is atranscription factor known to regulate a large number of gene productsinvolved in growth control. NF-kappaB-induced gene products includemolecules involved in immune, inflammatory, or acute phase responses,such as immunoglobulin light chain, major histocompatibility complex(MHC), IL-2R alpha chain, and cytokines such as IL-1beta, IL-6, andTNFalpha. NF-kappaB directly stimulates the HIV enhancer in T-cells andcan itself be activated by different viral proteins with oncogenicpotential, such as the hepatitis B virus HBX protein, EBV LMP1, andHTLV-1 Tax protein. The induction of NF-kappaB by Tax results inup-regulation of IL-2 and IL-2R and subsequently uncontrolled T-cellgrowth. IL-17 and HVS13, a gene product of HVS and a murine counterpartof IL-17, strongly induce IL-6 expression. IL-6 is a potent growthfactor for myelomas, plasmacytomas, and hybridomas and is involved inthe growth of Lennert's Lymphoma T-cells.

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc., FosterCity, Calif.), and all amino acid sequences of polypeptides encoded byDNA molecules determined herein were predicted by translation of a DNAsequence determined as above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

By “nucleotide sequence” of a nucleic acid molecule or polynucleotide isintended, for a DNA molecule or polynucleotide, a sequence ofdeoxyribonucleotides, and for an RNA molecule or polynucleotide, thecorresponding sequence of ribonucleotides (A, G, C and U), where eachthymidine deoxyribonucleotide (T) in the specified deoxyribonucleotidesequence is replaced by the ribonucleotide uridine (U).

Using the information provided herein, such as the nucleotide sequencein FIGS. 1A, 1B, and 1C (SEQ ED NO:1), a nucleic acid molecule of thepresent invention encoding a IL17RLP polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecule described in FIGS. 1A, 1B, and 1C (SEQ IDNO:1) was discovered in a cDNA library derived from human adultpulmonary tissue.

Additional clones of the same gene were also identified in cDNAlibraries from the following tissues: Crohn's Disease tissue, kidneypyramid, cortex, and medulla tissues, hippocampus, frontal cortex of thebrain from a patient with epilepsy, adrenal gland tumor, striatumdepression, osteclastoma, endometrial tumor, and hypothalamus from apatient with Schizophrenia.

The determined nucleotide sequence of the IL17RLP cDNA of FIGS. 1A, 1B,and 1C (SEQ ID NO:1) contains an open reading frame encoding a proteinof 426 amino acid residues, with an initiation codon at nucleotidepositions 10-12 of the nucleotide sequence in FIGS. 1A, 1B, and 1C (SEQID NO:1), and a deduced molecular weight of about 47.1 kDa. The aminoacid sequence of the IL17RLP protein shown in SEQ ID NO:2 is about 28.6%identical to the murine mRNA for IL-17 receptor (FIG. 2; Yao, Z., et al,Immunity 3:811-821 (1995); GenBank Accession No. U31993).

The open reading frame of the IL17RLP gene shares sequence homology withthe translation product of the murine mRNA for IL-17 receptor (FIG. 2;SEQ ID NO:3). The murine IL-17 receptor is thought to be important inregulation of immune cell signal transduction cascades and the resultingregulation of cell growth, differentiation, and activation-state. Thehomology between the murine IL-17 receptor and IL17RLP indicates thatIL17RLP may also be involved in regulation of immune cell signaltransduction cascades and the resulting regulation of cell growth,differentiation, and activation-state.

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors discussed above, the actual complete IL17RLPpolypeptide encoded by the deposited cDNA, which comprises about 426amino acids, may be somewhat longer or shorter. More generally, theactual open reading frame may be anywhere in the range of ±20 aminoacids, more likely in the range of ±10 amino acids, of that predictedfrom either the first methionine codon from the N-terminus shown inFIGS. 1A, 1B, and 1C (SEQ ID NO:1). It will further be appreciated that,depending on the analytical criteria used for identifying variousfunctional domains, the exact “address” of the extracellular,intracellular and transmembrane domains of the IL17RLP polypeptide maydiffer slightly from the predicted positions above. For example, theexact location of the IL17RLP extracellular domain in SEQ ID NO:2 mayvary slightly (e.g., the address may “shift” by about 1 to about 20residues, more likely about 1 to about 5 residues) depending on thecriteria used to define the domain. In this case, the ends of thetransmembrane domain and the beginning of the extracellular domain werepredicted on the basis of the identification of the hydrophobic aminoacid sequence in the above indicated positions, as shown in FIG. 3. Inany event, as discussed further below, the invention further providespolypeptides having various residues deleted from the N-terminus of thecomplete polypeptide, including polypeptides lacking one or more aminoacids from the N-terminus of the extracellular domain described herein,which constitute soluble forms of the extracellular domain of theIL17RLP protein.

In another embodiment, the present invention includes a polynucleotideof 1,918 nucleotides (SEQ ID NO:17) which encodes the IL17RLPpolypeptide provided in SEQ ID NO:18. The IL17RLP of SEQ ID NO:18differs from the IL17RLP provided in SEQ ID NO:2 only by the deletion ofthe C-terminal two residues (Cys-406 and Leu-407 of SEQ ID NO:2) and theaddition of nine amino acid residues (Leu-425 through Ile-433 of SEQ IDNO:18). The extracellular domain of IL17RLP is identical in SEQ ID NO:2and SEQ ID NO:18. The IL17RLP polynucleotide sequence shown in SEQ IDNO:17 was derived from sequencing the HAPOR40 cDNA clone deposited withthe ATCC™ with ATCC™ Deposit No. 209198 on Aug. 8, 1997.

It will further be appreciated that, depending on the analyticalcriteria used for identifying the exact location of the cleavage site ofthe precursor form of the mature IL17RLP molecule shown in SEQ ID NO:2may vary slightly, depending on the criteria used to define the cleavagesite. In this case, the ends of the signal peptide and the beginning ofthe mature IL17RLP molecule were predicted using the HGSI SignalPcomputer algorithm. One of skill in the art will realize that anotherwidely accepted computer algorithm used to predict potential sites ofpolypeptide cleavage, PSORT, will predict the cleavage of an N-terminalsignal peptide from the IL17RLP polypeptide at a point slightlydifferent from that predicted by the HGSI SignalP algorithm. In eithercase, as discussed further below, the invention further providespolypeptides having various residues deleted from the N-terminus of thecomplete polypeptide, including polypeptides corresponding to either ofthe predicted mature IL17RLP polypeptides described herein.

Leader and Mature Sequences

The amino acid sequence of the complete IL17RLP protein includes aleader sequence and a mature protein, as shown in SEQ ID NO:2. More inparticular, the present invention provides nucleic acid moleculesencoding a mature form of the IL17RLP protein. Thus, according to thesignal hypothesis, once export of the growing protein chain across therough endoplasmic reticulum has been initiated, proteins secreted bymammalian cells have a signal or secretory leader sequence which iscleaved from the complete polypeptide to produce a secreted “mature”form of the protein. Most mammalian cells and even insect cells cleavesecreted proteins with the same specificity. However, in some cases,cleavage of a secreted protein is not entirely uniform, which results intwo or more mature species of the protein. Further, it has long beenknown that the cleavage specificity of a secreted protein is ultimatelydetermined by the primary structure of the complete protein, that is, itis inherent in the amino acid sequence of the polypeptide. Therefore,the present invention provides a nucleotide sequence encoding the matureIL17RLP polypeptide having the amino acid sequence encoded by the cDNAclone identified as ATCC™ Deposit No. 209198. By the “mature IL17RLPpolypeptide having the amino acid sequence encoded by the cDNA clone inATCC™ Deposit No. 209198” is meant the mature form(s) of the IL17RLPprotein produced by expression in a mammalian cell (e.g., COS cells, asdescribed below) of the complete open reading frame encoded by the humanDNA sequence of the clone contained in the deposited clone HAPOR40.

In addition, methods for predicting whether a protein has a secretoryleader as well as the cleavage point for that leader sequence areavailable. For instance, the method of McGeoch (Virus Res. 3:271-286(1985)) uses the information from a short N-terminal charged region anda subsequent uncharged region of the complete (uncleaved) protein. Themethod of von Heinje (Nucleic Acids Res. 14:4683-4690 (1986)) uses theinformation from the residues surrounding the cleavage site, typicallyresidues −13 to +2 where +1 indicates the amino terminus of the matureprotein. The accuracy of predicting the cleavage points of knownmammalian secretory proteins for each of these methods is in the rangeof 75-80% (von Heinje, supra). However, the two methods do not alwaysproduce the same predicted cleavage point(s) for a given protein.

In the present case, the deduced amino acid sequence of the completeIL17RLP polypeptide was analyzed by a variation of the computer program“PSORT”, available from Dr. Kenta Nakai of the Institute for ChemicalResearch, Kyoto University (Nakai, K. and Kanehisa, M. Genomics14:897-911 (1992)), which is an expert system for predicting thecellular location of a protein based on the amino acid sequence. As partof this computational prediction of localization, the methods of McGeochand von Heinje are incorporated. Thus, the computation analysis abovepredicted a single cleavage site within the complete amino acid sequenceshown in SEQ ID NO:2 (see above discussion).

As one of ordinary skill would appreciate from the above discussions,due to the possibilities of sequencing errors as well as the variabilityof cleavage sites in different known proteins, the mature IL17RLPpolypeptide encoded by the deposited cDNA is expected to consist ofabout 407 amino acids (presumably residues 1 to 407 of SEQ ID NO:2, butmay consist of any number of amino acids in the range of about 407-412amino acids (e.g., 407, 408, 409, 410, 411, and/or 412); and the actualleader sequence(s) of this protein is expected to be 14-19 amino acids(presumably residues −19 through −1 of SEQ ID NO:2), but may consist ofany number of amino acids in the range of 14-19 amino acids (e.g., 14,15, 16, 17, 18 and/or 19).

In another embodiment, the encoded polypeptide has a predicted leadersequence from Met-(−19) to Ser-(−6) of SEQ ID NO:2 (i.e., from Met-1 toSer-14 of the amino acid sequence presented in FIGS. 1A, 1B, and 1C); anextracellular domain from Ala-(−5) to Trp-271 of SEQ ID NO:2 (i.e., fromAla-15 to Trp-290 of the amino acid sequence presented in FIGS. 1A, 1B,and 1C); a transmembrane domain from Leu-272 to Leu-292 of SEQ ID NO:2(i.e., from Leu-291 to Leu-311 of the amino acid sequence presented inFIGS. 1A, 1B, and 1C); and an intracellular domain from Met-293 toLeu-407 of SEQ ID NO:2 (i.e., from Met-312 to Leu-426 of the amino acidsequence presented in FIGS. 1A, 1B, and 1C). The predicted leaderpeptide in this embodiment is within the originally predicted range of14-19 amino acids.

In an additional embodiment, the IL17RLP transmembrane domain may havean N-terminal boundary beginning at amino acid residue Pro-268, Gly-269,Gly-270, Trp-271 or Leu-272 of the IL17RLP sequence as shown in SEQ IDNO:2 (i.e., amino acid residues Pro-287, Gly-288, Gly-289, Trp-290 orLeu-291 of the IL17RLP sequence as shown in FIGS. 1A, 1B, and 1C) and aC-terminal boundary including amino acid residue Tyr-291, Leu-292,Met-293 or Trp-294 of the IL17RLP sequence as shown in SEQ ID NO:2(i.e., amino acid residues Tyr-310, Leu-311, Met-312 or Trp-313 of theIL17RLP sequence as shown in FIGS. 1A, 1B, and 1C).

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically. However, a nucleic acid contained in aclone that is a member of a library (e.g., a genomic or cDNA library)that has not been isolated from other members of the library (e.g., inthe form of a homogeneous solution containing the clone and othermembers of the library) or which is contained on a chromosomepreparation (e.g., a chromosome spread) or a nucleic acid present in apreparation of genomic DNA (e.g., intact, sheared, and/or cut with oneor more restriction enzymes) that has not been isolated from othernucleic acids in the preparation, is not “isolated” for the purposes ofthis invention.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising an open reading frame (ORF) with an initiationcodon at positions 10-12 of the nucleotide sequence shown in FIGS. 1A,1B, and 1C (SEQ ID NO:1).

Also included are DNA molecules comprising the coding sequence for thepredicted mature IL17RLP protein shown at positions 1-407 of SEQ IDNO:2.

In addition, isolated nucleic acid molecules of the invention includeDNA molecules which comprise a sequence substantially different fromthose described above but which, due to the degeneracy of the geneticcode, still encode the IL17RLP protein. Of course, the genetic code andspecies-specific codon preferences are well known in the art. Thus, itwould be routine for one skilled in the art to generate the degeneratevariants described above, for instance, to optimize codon expression fora particular host (e.g., change codons in the human mRNA to thosepreferred by a bacterial host such as E. coli).

In another aspect, the invention provides isolated nucleic acidmolecules encoding the IL17RLP polypeptide having an amino acid sequenceencoded by the cDNA clone contained in the plasmid deposited as ATCC™Deposit No. 209198 on Aug. 8, 1997.

Preferably, this nucleic acid molecule will encode the maturepolypeptide encoded by the above-described deposited cDNA clone. Alsopreferably, this nucleic acid molecule will encode the extracellulardomain encoded by the above-described cDNA clone.

The invention further provides an isolated nucleic acid molecule havingthe nucleotide sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) orthe nucleotide sequence of the IL17RLP cDNA contained in theabove-described deposited clone, or a nucleic acid molecule having asequence complementary to one of the above sequences. Such isolatedmolecules, particularly DNA molecules, are useful as probes for genemapping, by in situ hybridization with chromosomes, and for detectingexpression of the IL17RLP gene in human tissue, for instance, byNorthern blot analysis.

The present invention is further directed to nucleic acid moleculesencoding portions of the nucleotide sequences described herein as wellas to fragments of the isolated nucleic acid molecules described herein.In particular, the invention provides a polynucleotide having anucleotide sequence representing the portion of SEQ ID NO:1 whichconsists of positions 1-1290 of SEQ ID NO:1.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:1 whichhave been determined from the following related cDNA clones: HHPCH63R(SEQ ID NO:4) and HETCC45RA (SEQ ID NO:5). Such polynucleotides maypreferably be excluded from the invention.

Further, the invention includes a polynucleotide comprising, oralternatively consisting of, any portion of at least about 25nucleotides, at least about 30 nucleotides, at least about 35nucleotides, at least about 40 nucleotides, at least about 45nucleotides, preferably at least about 50 nucleotides, at least about 60nucleotides, at least about 70 nucleotides, at least about 80nucleotides, at least about 90 nucleotides, or at least about 100nucleotides of SEQ ID NO:1 from residue 50-1800, 100-1800, 200-1800,300-1800, 400-1800, 500-1800, 600-1800, 50-650, 100-650, 200-650,300-650, 400-650, 500-650, 50-500, 100-500, 200-500, 300-500, 400-500,50-400, 100-400, 200-400, 300-400, 50-300, 100-300, 200-300, 50-200,100-200, and 50-100.

More generally, by a fragment of an isolated nucleic acid moleculehaving the nucleotide sequence of the deposited cDNA or the nucleotidesequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) is intendedfragments at least about 15 nt, and more preferably at least about 20nt, more preferably at least about 25 nt, still more preferably at leastabout 30 nt, more preferably at least about 35 nt, and even morepreferably, at least about 40 nt, more preferably at least about 45 nt,in length which are useful as diagnostic probes and primers as discussedherein. Of course, larger fragments 50-300 nt in length (e.g., 50 nt, 55nt, 60 nt, 65 nt, 70 nt, 75 nt, 80 nt, 85 nt, 90 nt, 95 nt, 100 nt, 125nt, 150 nt, 175 nt, 200 nt, 225 nt, 250 nt, 275 nt, and/or 300 nt (ofcourse, fragment lengths in addition to those recited herein are alsouseful)) are also useful according to the present invention as arefragments corresponding to most, if not all, of the nucleotide sequenceof the deposited cDNA or as shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1).By a fragment at least 20 nt in length, for example, is intendedfragments which include 20 or more contiguous bases from the nucleotidesequence of the deposited cDNA or the nucleotide sequence as shown inFIGS. 1A, 1B, and 1C (SEQ ID NO:1). Preferred nucleic acid fragments ofthe present invention include nucleic acid molecules encodingepitope-bearing portions of the IL17RLP polypeptide as identified, forexample, in FIG. 3 and described in more detail below.

In specific embodiments, the polynucleotide fragments of the inventionencode a polypeptide which demonstrates a functional activity. By apolypeptide demonstrating “functional activity” is meant, a polypeptidecapable of displaying one or more known functional activities associatedwith a complete, mature, extracellular domain, or active form of theIL17RLP polypeptide. Such functional activities include, but are notlimited to, biological activity ((e.g., activation of signaltransduction pathways resulting in the stimulation of the NF-kappaBtranscription factor family, the secretion of IL-6, and thecostimulation of T-cell proliferation; induction of IL-6, IL-8, G-CSF,Prostaglandin E (PGE₂), and intracellular adhesion molecule (ICAM)-1expression; regulation of hematopoietic stem and progenitor cell growthand expansion; myelosuppressive activity for stem and immature subsetsof myeloid progenitors; activation and stimulation of hematopoiesis ingeneral (more specifically, neutrophil hematopoiesis); enhancement oferythropoiesis; suppression of lymphopoiesis and myelopoiesis; andstrong suppression of monocytopoiesis)), antigenicity [ability to bind(or compete with a IL17RLP polypeptide for binding) to an anti-IL17RLPantibody], immunogenicity (ability to generate antibody which binds toan IL17RLP polypeptide), the ability to form polymers with other IL17RLPor IL17RLP-like polypeptides, and ability to bind to a receptor orligand for an IL17RLP polypeptide.

Preferred nucleic acid fragments of the present invention also includenucleic acid molecules encoding polypeptides comprising, oralternatively consisting of, one or more of the following domains ofIL17RLP: Domain I (i.e., Val-49 through Leu-62 of SEQ ID NO:2 (Val-68through Leu-81 of FIGS. 1A, 1B, and 1C)); Domain II (Cys-154 throughThr-166 of SEQ ID NO:2 (i.e., Cys-173 through Thr-185 of FIGS. 1A, 1B,and 1C)); Domain III (Gln-202 through Gln-208 of SEQ ID NO:2 (i.e.,Gln-221 through Gln-227 of FIGS. 1A, 1B, and 1C)); Domain IV (Asp-241through Val-249 of SEQ ID NO:2 (i.e., Asp-260 through Val-268 of FIGS.1A, 1B, and 1C)); Domain V (Thr-255 through Leu-261 of SEQ ID NO:2(i.e., Thr-274 through Leu-280 of FIGS. 1A, 1B, and 1C)); Domain VI(Leu-310 through Tyr-319 of SEQ ID NO:2 (i.e., Leu-329 through Tyr-338of FIGS. 1A, 1B, and 1C)); Domain VII (Cys-340 through Leu-346 of SEQ IDNO:2 (i.e., Cys-359 through Leu-365 of FIGS. 1A, 1B, and 1C)); andDomain VIII (Ile-354 through Gly-358 of SEQ ID NO:2 (i.e., Ile-373through Gly-377 of FIGS. 1A, 1B, and 1C)).

In specific embodiments, the polynucleotide fragments of the inventionencode antigenic regions. Non-limiting examples of antigenicpolypeptides or peptides that can be used to generate IL17RLP-specificantibodies include: a polypeptide comprising, or alternativelyconsisting of, amino acid residues from about from about Ser-14 to aboutVal-22, from about Cys-24 to about Pro-32, from about Ile-41 to aboutArg-49, from about Thr-89 to about, from about Thr-110 to about Lys-118,from about Ala-144 to about Ser-152, from about Thr-240 to aboutVal-248, from about Gly-258 to about Thr-267, from about Leu-280 toabout Gly-288, from about Cys-404 to about Glu-412, from about Pro-415to about Ser-423, from about Gly-409 to about Glu-417, and from aboutCys-404 to about Leu-426 in FIGS. 1A, 1B, and 1C (which is the identicalsequence to that shown in SEQ ID NO:2, with the exception of thenumbering schemes as described above).

Additional preferred polypeptide fragments comprise, or alternativelyconsist of, the amino acid sequence of residues: M-1 to A-15; S-2 toV-16; L-3 to P-17; V-4 to R-18; L-5 to E-19; L-6 to P-20; S-7 to T-21;L-8 to V-22; A-9 to Q-23; A-10 to C-24; L-11 to G-25; C-12 to S-26; R-13to E-27; S-14 to T-28; A-15 to G-29; V-16 to P-30; P-17 to S-31; R-18 toP-32; E-19 to E-33; P-20 to W-34; T-21 to M-35; V-22 to L-36; Q-23 toQ-37; C-24 to H-38; G-25 to D-39; S-26 to L-40; E-27 to I-41; T-28 toP-42; G-29 to G-43; P-30 to D-44; S-31 to L-45; P-32 to R-46; E-33 toD-47; W-34 to L-48; M-35 to R-49; L-36 to V-50; Q-37 to E-51; H-38 toP-52; D-39 to V-53; L-40 to T-54; I-41 to T-55; P-42 to S-56; G-43 toV-57; D-44 to A-58; L-45 to T-59; R-46 to G-60; D-47 to D-61; L-48 toY-62; R-49 to S-63; V-50 to I-64; E-51 to L-65; P-52 to M-66; V-53 toN-67; T-54 to V-68; T-55 to S-69; S-56 to W-70; V-57 to V-71; A-58 toL-72; T-59 to R-73; G-60 to A-74; D-61 to D-75; Y-62 to A-76; S-63 toS-77; I-64 to I-78; L-65 to R-79; M-66 to L-80; N-67 to L-81; V-68 toK-82; S-69 to A-83; W-70 to T-84; V-71 to K-85; L-72 to I-86; R-73 toC-87; A-74 to V-88; D-75 to T-89; A-76 to G-90; S-77 to K-91; I-78 toS-92; R-79 to N-93; L-80 to F-94; L-81 to Q-95; K-82 to S-96; A-83 toY-97; T-84 to S-98; K-85 to C-99; I-86 to V-100; C-87 to R-101; V-88 toC-102; T-89 to N-103; G-90 to Y-104; K-91 to T-105; S-92 to E-106; N-93to A-107; F-94 to F-108; Q-95 to Q-109; S-96 to T-110; Y-97 to Q-111;S-98 to T-112; C-99 to R-113; V-100 to P-114; R-101 to S-115; C-102 toG-116; N-103 to G-117; Y-104 to K-118; T-105 to W-119; E-106 to T-120;A-107 to F-121; F-108 to S-122; Q-109 to Y-123; T-110 to I-124; Q-111 toG-125; T-112 to F-126; R-113 to P-127; P-114 to V-128; S-115 to E-129;G-116 to L-130; G-117 to N-131; K-118 to T-132; W-119 to V-133; T-120 toY-134; F-121 to F-135; S-122 to I-136; Y-123 to G-137; I-124 to A-138;G-125 to H-139; F-126 to N-140; P-127 to I-141; V-128 to P-142; E-129 toN-143; L-130 to A-144; N-131 to N-145; T-132 to M-146; V-133 to N-147;Y-134 to E-148; F-135 to D-149; I-136 to G-150; G-137 to P-151; A-138 toS-152; H-139 to M-153; N-140 to S-154; I-141 to V-155; P-142 to N-156;N-143 to F-157; A-144 to T-158; N-145 to S-159; M-146 to P-160; N-147 toG-161; E-148 to C-162; D-149 to L-163; G-150 to D-164; P-151 to H-165;S-152 to I-166; M-153 to M-167; S-154 to K-168; V-155 to Y-169; N-156 toK-170; F-157 to K-171; T-158 to K-172; S-159 to C-173; P-160 to V-174;G-161 to K-175; C-162 to A-176; L-163 to G-177; D-164 to S-178; H-165 toL-179; I-166 to W-180; M-167 to D-181; K-168 to P-182; Y-169 to N-183;K-170 to I-184; K-171 to T-185; K-172 to A-186; C-173 to C-187; V-174 toK-188; K-175 to K-189; A-176 to N-190; G-177 to E-191; S-178 to E-192;L-179 to T-193; W-180 to V-194; D-181 to E-195; P-182 to V-196; N-183 toN-197; I-184 to F-198; T-185 to T-199; A-186 to T-200; C-187 to T-201;K-188 to P-202; K-189 to L-203; N-190 to G-204; E-191 to N-205; E-192 toR-206; T-193 to Y-207; V-194 to M-208; E-195 to A-209; V-196 to L-210;N-197 to I-211; F-198 to Q-212; T-199 to H-213; T-200 to S-214; T-201 toT-215; P-202 to I-216; L-203 to I-217; G-204 to G-218; N-205 to F-219;R-206 to S-220; Y-207 to Q-221; M-208 to V-222; A-209 to F-223; L-210 toE-224; I-211 to P-225; Q-212 to H-226; H-213 to Q-227; S-214 to K-228;T-215 to K-229; I-216 to Q-230; I-217 to T-231; G-218 to R-232; F-219 toA-233; S-220 to S-234; Q-221 to V-235; V-222 to V-236; F-223 to I-237;E-224 to P-238; P-225 to V-239; H-226 to T-240; Q-227 to G-241; K-228 toD-242; K-229 to S-243; Q-230 to E-244; T-231 to G-245; R-232 to A-246;A-233 to T-247; S-234 to V-248; V-235 to Q-249; V-236 to L-250; I-237 toT-251; P-238 to P-252; V-239 to Y-253; T-240 to F-254; G-241 to P-255;D-242 to T-256; S-243 to C-257; E-244 to G-258; G-245 to S-259; A-246 toD-260; T-247 to C-261; V-248 to I-262; Q-249 to R-263; L-250 to H-264;T-251 to K-265; P-252 to G-266; Y-253 to T-267; F-254 to V-268; P-255 toV-269; T-256 to L-270; C-257 to C-271; G-258 to P-272; S-259 to Q-273;D-260 to T-274; C-261 to G-275; I-262 to V-276; R-263 to P-277; H-264 toF-278; K-265 to P-279; G-266 to L-280; T-267 to D-281; V-268 to N-282;V-269 to N-283; L-270 to K-284; C-271 to S-285; P-272 to K-286; Q-273 toP-287; T-274 to G-288; G-275 to G-289; V-276 to W-290; P-277 to L-291;F-278 to P-292; P-279 to L-293; L-280 to L-294; D-281 to L-295; N-282 toL-296; N-283 to S-297; K-284 to L-298; S-285 to L-299; K-286 to V-300;P-287 to A-301; G-288 to T-302; G-289 to W-303; W-290 to V-304; L-291 toL-305; P-292 to V-306; L-293 to A-307; L-294 to G-308; L-295 to I-309;L-296 to Y-310; S-297 to L-311; L-298 to M-312; L-299 to W-313; V-300 toR-314; A-301 to H-315; T-302 to E-316; W-303 to R-317; V-304 to I-318;L-305 to K-319; V-306 to K-320; A-307 to T-321; G-308 to S-322; I-309 toF-323; Y-310 to S-324; L-311 to T-325; M-312 to T-326; W-313 to T-327;R-314 to L-328; H-315 to L-329; E-316 to P-330; R-317 to P-331; I-318 toI-332; K-319 to K-333; K-320 to V-334; T-321 to L-335; S-322 to V-336;F-323 to V-337; S-324 to Y-338; T-325 to P-339; T-326 to S-340; T-327 toE-341; L-328 to I-342; L-329 to C-343; P-330 to F-344; P-331 to H-345;I-332 to H-346; K-333 to T-347; V-334 to I-348; L-335 to C-349; V-336 toY-350; V-337 to F-351; Y-338 to T-352; P-339 to E-353; S-340 to F-354;E-341 to L-355; I-342 to Q-356; C-343 to N-357; F-344 to H-358; H-345 toC-359; H-346 to R-360; T-347 to S-361; I-348 to E-362; C-349 to V-363;Y-350 to I-364; F-351 to L-365; T-352 to E-366; E-353 to K-367; F-354 toW-368; L-355 to Q-369; Q-356 to K-370; N-357 to K-371; H-358 to K-372;C-359 to I-373; R-360 to A-374; S-361 to E-375; E-362 to M-376; V-363 toG-377; I-364 to P-378; L-365 to V-379; E-366 to Q-380; K-367 to W-381;W-368 to L-382; Q-369 to A-383; K-370 to T-384; K-371 to Q-385; K-372 toK-386; I-373 to K-387; A-374 to A-388; E-375 to A-389; M-376 to D-390;G-377 to K-391; P-378 to V-392; V-379 to V-393; Q-380 to F-394; W-381 toL-395; L-382 to L-396; A-383 to S-397; T-384 to N-398; Q-385 to D-399;K-386 to V-400; K-387 to N-401; A-388 to S-402; A-389 to V-403; D-390 toC-404; K-391 to D-405; V-392 to G-406; V-393 to T-407; F-394 to C-408;L-395 to 0-409; L-396 to K-410; S-397 to S-411; N-398 to E-412; D-399 toG-413; V-400 to S-414; N-401 to P-415; S-402 to S-416; V-403 to E-417;C-404 to N-418; D-405 to S-419; G-406 to Q-420; I-407 to D-421; C-408 toS-422; G-409 to S-423; K-410 to P-424; S-411 to C-425; and E-412 toL-426 of the amino acid sequence in shown in SEQ ID NO:2. Thesepolypeptide fragments may retain the biological activity of IL17RLPpolypeptides of the invention and/or may be useful to generate or screenfor antibodies, as described further below. Polynucleotides encodingthese polypeptide fragments are also encompassed by the invention.

Preferably, the polynucleotide fragments of the invention encode apolypeptide which demonstrates a IL17RLP functional activity. By apolypeptide demonstrating a IL17RLP “functional activity” is meant, apolypeptide capable of displaying one or more known functionalactivities associated with a full-length (complete) IL17RLP protein.Such functional activities include, but are not limited to, biologicalactivity, antigenicity [ability to bind (or compete with a IL17RLPpolypeptide for binding) to an anti-IL17RLP antibody], immunogenicity(ability to generate antibody which binds to a IL17RLP polypeptide),ability to form multimers with IL17RLP polypeptides of the invention,and ability to bind to a receptor or ligand for a IL17RLP polypeptide.

The functional activity of IL17RLP polypeptides, and fragments, variantsderivatives, and analogs thereof, can be assayed by various methods.

In additional embodiments, the polynucleotides of the invention encodefunctional attributes of IL17RLP. Preferred embodiments of the inventionin this regard include fragments that comprise, or alternatively consistof, alpha-helix and alpha-helix forming regions (“alpha-regions”),beta-sheet and beta-sheet forming regions (“beta-regions”), turn andturn-forming regions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions and high antigenic index regions of IL17RLP.

The data representing the structural or functional attributes of IL17RLPset forth in FIG. 3 and/or Table I, as described above, was generatedusing the various modules and algorithms of the DNA*STAR set on defaultparameters. In a preferred embodiment, the data presented in columnsVIII, IX, XIII, and XIV of Table I can be used to determine regions ofIL17RLP which exhibit a high degree of potential for antigenicity.Regions of high antigenicity are determined from the data presented incolumns VIII, IX, XIII, and/or IV by choosing values which representregions of the polypeptide which are likely to be exposed on the surfaceof the polypeptide in an environment in which antigen recognition mayoccur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in FIG. 3, butmay, as shown in Table I, be represented or identified by using tabularrepresentations of the data presented in FIG. 3. The DNA*STAR computeralgorithm used to generate FIG. 3 (set on the original defaultparameters) was used to present the data in FIG. 3 in a tabular format(See Table I). The tabular format of the data in FIG. 3 may be used toeasily determine specific boundaries of a preferred region.

The above-mentioned preferred regions set out in FIG. 3 and in Table Iinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIGS. 1A,1B, and 1C. As set out in FIG. 3 and in Table I, such preferred regionsinclude Garnier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions,Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenbergalpha- and beta-amphipathic regions, Karplus-Schulz flexible regions,Emini surface-forming regions and Jameson-Wolf regions of high antigenicindex.

TABLE I Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met1 A A . . . . . −1.43 0.61 . . . −0.60 0.30 Ser 2 A A . . . . . −1.860.87 . . . −0.60 0.20 Leu 3 A A . . . . . −1.77 1.13 . . . −0.60 0.13Val 4 A A . . . . . −2.19 1.09 . . . −0.60 0.17 Leu 5 A A . . . . .−2.39 1.16 . . . −0.60 0.11 Leu 6 A A . . . . . −2.38 1.27 . . . −0.600.13 Ser 7 A A . . . . . −2.89 1.09 . . . −0.60 0.18 Leu 8 A A . . . . .−2.74 1.13 * * . −0.60 0.18 Ala 9 A A . . . . . −1.78 1.01 * * . −0.600.11 Ala 10 A A . . . . . −1.27 0.33 * . . −0.30 0.17 Leu 11 A A . . . .. −1.04 0.33 * * . −0.30 0.27 Cys 12 A . B . . T . −1.60 0.14 * . . 0.100.27 Arg 13 . . B . . T . −1.00 0.29 * . . 0.40 0.20 Ser 14 . . B . . T. −0.30 0.21 . . . 0.70 0.37 Ala 15 . . B . . T . 0.29 −0.47 . . . 1.751.37 Val 16 . . . . . . C 0.89 −1.04 * . F 2.50 1.21 Pro 17 . . . . T .. 1.24 −0.61 * . F 3.00 1.39 Arg 18 . . . . T . . 0.28 −0.51 . . F 2.701.99 Glu 19 . . B . . . . 0.58 −0.37 . . F 1.70 1.99 Pro 20 . . B . . .. 0.50 −0.61 . . F 1.70 2.23 Thr 21 . . B . . . . 1.01 −0.47 . . F 0.950.61 Val 22 . . B . . . . 0.92 −0.04 . . . 0.50 0.35 Gln 23 . . B . . .. 0.81 0.34 . . . 0.18 0.30 Cys 24 . . B . . T . 0.50 −0.09 * . F 1.410.36 Gly 25 . . B . . T . 0.37 −0.09 * . F 1.69 0.71 Ser 26 . . . . T T. 0.47 −0.30 * . F 2.37 0.40 Glu 27 . . . . T T . 1.02 −0.27 * . F 2.801.16 Thr 28 . . . . . . C 0.81 −0.46 * . F 2.12 1.57 Gly 29 . . . . . .C 1.48 −0.46 . . F 1.84 1.82 Pro 30 . . . . . . C 1.53 −0.84 . . F 1.861.82 Ser 31 . . . . . T C 1.23 0.07 . . F 0.88 1.32 Pro 32 . . . . . T C0.42 0.20 . * F 0.60 1.32 Glu 33 A . . . . T . 0.73 0.46 . . F −0.050.71 Trp 34 A . . . . T . 1.04 0.43 . * . −0.20 0.91 Met 35 A A . . . .. 1.26 0.54 . * . −0.60 0.80 Leu 36 A A . . . . . 0.74 0.11 . * . −0.300.77 Gln 37 A A . . . . . 0.07 0.80 . * . −0.60 0.61 His 38 . A B . . .. −0.14 0.57 . * . −0.60 0.43 Asp 39 . A . . T . . −0.20 0.39 . . . 0.100.81 Leu 40 . A . . . . C 0.40 0.13 . . . 0.24 0.46 Ile 41 . . B . . T .0.40 −0.27 * * . 1.38 0.57 Pro 42 . . B . . T . 0.51 −0.09 * * F 1.870.28 Gly 43 . . . . T T . 0.54 −0.09 * * F 2.61 0.66 Asp 44 . . . . T T. −0.27 −0.77 * . F 3.40 1.58 Leu 45 . A B . . . . 0.66 −0.77 * * F 2.110.84 Arg 46 . A B . . . . 0.69 −1.20 * * F 1.92 1.67 Asp 47 . A B . . .. 0.90 −0.99 . * F 1.43 0.74 Leu 48 . A B . . . . 1.03 −0.99 . . . 1.091.56 Arg 49 . A B . . . . 0.18 −1.24 . . . 0.75 1.23 Val 50 . A B B . .. 0.68 −0.60 * . . 0.60 0.55 Glu 51 . A B B . . . 0.26 −0.11 * * F 0.450.96 Pro 52 . A B B . . . −0.04 −0.31 . * F 0.45 0.70 Val 53 . . B B . .. −0.09 0.07 * * F 0.00 1.27 Thr 54 . . B B . . . −0.79 0.07 * * F −0.150.55 Thr 55 . . B B . . . −0.24 0.57 * . F −0.45 0.36 Ser 56 . . B B . .. −0.59 0.63 * . F −0.45 0.69 Val 57 . . B B . . . −0.38 0.41 . . F−0.45 0.47 Ala 58 . . B B . . . 0.23 −0.07 . . F 0.45 0.55 Thr 59 . . B. . T . 0.24 0.20 . * F 0.25 0.64 Gly 60 . . B . . T . −0.33 0.20 . . F0.40 1.16 Asp 61 . . B . . T . −0.84 0.24 . . F 0.25 0.80 Tyr 62 . . B .. T . −0.59 0.43 . * . −0.20 0.46 Ser 63 . . B B . . . −0.00 0.56 . * .−0.60 0.46 Ile 64 . . B B . . . −0.54 0.53 . * . −0.60 0.44 Leu 65 . . BB . . . −0.50 1.17 . * . −0.60 0.21 Met 66 . . B B . . . −0.79 0.80 . *. −0.60 0.21 Asn 67 . . B B . . . −1.40 1.33 * * . −0.60 0.31 Val 68 . .B B . . . −1.91 1.29 * * . −0.60 0.28 Ser 69 . . B B . . . −0.911.29 * * . −0.60 0.24 Trp 70 . . B B . . . −0.69 0.67 * * . −0.60 0.29Val 71 . . B B . . . −0.09 0.77 . * . −0.60 0.39 Leu 72 A . . B . . .−0.68 0.13 * * . −0.30 0.49 Arg 73 A . . B . . . −0.12 0.24 * * . −0.300.47 Ala 74 A . . B . . . −0.71 −0.29 * * . 0.30 0.85 Asp 75 A . . B . .. −0.31 −0.24 * * . 0.30 0.72 Ala 76 A . . B . . . −0.27 −0.93 * * .0.60 0.72 Ser 77 A . . B . . . −0.27 −0.24 * * . 0.30 0.59 Ile 78 A . .B . . . −0.33 −0.06 * * . 0.30 0.29 Arg 79 A . . B . . . −0.33 −0.06 * *. 0.30 0.57 Leu 80 A . . B . . . −0.64 −0.06 * * . 0.30 0.43 Leu 81 A .. B . . . −0.01 0.04 * * . −0.30 0.89 Lys 82 A . . B . . . −0.60−0.64 * * F 0.75 0.91 Ala 83 A . . B . . . −0.38 0.04 * * F −0.15 0.77Thr 84 A . . B . . . −1.34 −0.07 * . F 0.45 0.50 Lys 85 . . B B . . .−0.84 −0.11 * . F 0.45 0.19 Ile 86 . . B B . . . −0.38 0.37 . * . −0.300.27 Cys 87 . . B B . . . −0.38 0.30 * * . −0.30 0.18 Val 88 . . B B . .. −0.09 −0.19 * * . 0.58 0.18 Thr 89 . . B B . . . 0.22 0.20 * * F 0.410.35 Gly 90 . . . . T T . −0.52 −0.09 * * F 2.24 1.05 Lys 91 . . . . T T. 0.37 0.13 * * F 1.92 1.22 Ser 92 . . . . T T . 0.73 −0.11 . * F 2.801.47 Asn 93 . . . . T T . 1.34 −0.21 . * F 2.52 1.99 Phe 94 . . . . T .. 1.36 0.11 . * F 1.44 1.56 Gln 95 . . . . T . . 1.03 0.50 * * F 0.861.56 Ser 96 . . . . T T . 0.13 0.69 * * . 0.48 0.52 Tyr 97 . . B . . T .0.54 0.93 . * . −0.20 0.44 Ser 98 . . . . T T . −0.12 0.14 . * . 0.500.50 Cys 99 . . B . . T . 0.58 0.31 . * . 0.10 0.20 Val 100 . . B . . .. 0.33 0.33 . * . 0.12 0.21 Arg 101 . . B . . . . 0.32 0.33 . * . 0.340.24 Cys 102 . . B . . T . 0.57 0.43 * * . 0.46 0.65 Asn 103 . . . . T T. 0.28 −0.14 * * . 2.13 1.52 Tyr 104 . . . . T T . 0.24 −0.29 * * . 2.200.78 Thr 105 . . . . . T C 1.10 0.50 * * . 1.03 1.26 Glu 106 . A B B . .. 0.68 0.33 . * . 0.51 1.36 Ala 107 . A B B . . . 1.34 0.41 . . . −0.011.25 Phe 108 . A B B . . . 1.03 0.06 * * F 0.22 1.50 Gln 109 . A B B . .. 1.39 0.06 * * F 0.00 1.25 Thr 110 . A B B . . . 1.49 0.06 * * F 0.002.43 Gln 111 . . B B . . . 1.19 −0.01 * * F 0.94 4.34 Thr 112 . . . B .. C 1.43 −0.41 * * F 1.48 3.36 Arg 113 . . . B . . C 1.79 −0.39 . * F1.82 2.30 Pro 114 . . . . T T . 1.83 −0.44 . * F 2.76 1.32 Ser 115 . . .. T T . 1.86 −0.84 . * F 3.40 1.82 Gly 116 . . . . T T . 1.54 −0.41 . *F 2.61 0.98 Gly 117 . . . . T T . 1.16 0.07 . * F 1.67 0.91 Lys 118 . .. B T . . 0.74 0.43 . * F 0.63 0.59 Trp 119 . . B B . . . 0.71 0.43 * .. −0.26 0.80 Thr 120 . . B B . . . 0.12 0.76 * * . −0.45 1.26 Phe 121 .. B B . . . 0.12 1.01 . * . −0.60 0.44 Ser 122 . . B B . . . −0.23 1.44. * . −0.60 0.42 Tyr 123 . . B B . . . −0.49 1.31 . * . −0.60 0.25 Ile124 . . . B T . . −1.06 1.26 . * . −0.20 0.45 Gly 125 . . . B . . C−0.74 1.11 . * . −0.40 0.25 Phe 126 . . . B . . C −0.86 0.73 . * . −0.400.27 Pro 127 . . B . . . . −0.56 0.66 . * . −0.40 0.32 Val 128 . . B . .. . −0.62 0.37 . * . −0.10 0.52 Glu 129 . . B . . . . −0.59 0.43 . * .−0.40 0.87 Leu 130 . . B B . . . −0.49 0.29 . * . −0.30 0.42 Asn 131 . .B B . . . −0.49 0.61 . * . −0.60 0.88 Thr 132 . . B B . . . −1.17 0.76 .. . −0.60 0.44 Val 133 . . B B . . . −0.66 1.44 . . . −0.60 0.38 Tyr 134. . B B . . . −1.24 1.19 . . . −0.60 0.23 Phe 135 . . B B . . . −0.471.29 . . . −0.60 0.16 Ile 136 . . B B . . . −0.47 1.30 . . . −0.60 0.30Gly 137 . . B . . . . −1.04 1.06 . . . −0.40 0.30 Ala 138 . . B . . . .−0.40 0.99 . . . −0.40 0.25 His 139 . . . . . . C −0.16 0.63 * . . −0.200.54 Asn 140 . . . . . . C −0.04 0.34 . . . 0.10 0.88 Ile 141 . . . . .T C 0.84 0.41 . . . 0.00 0.88 Pro 142 . . . . . T C 0.59 0.31 * . F 0.601.04 Asn 143 . . . . T T . 1.18 0.43 * . F 0.35 0.64 Ala 144 . . . . . TC 1.21 0.43 . . F 0.64 1.47 Asn 145 . . . . . . C 1.21 −0.26 . * . 1.531.65 Met 146 . . B . . . . 1.76 −0.69 . * . 1.97 1.71 Asn 147 . . . . .T C 1.76 −0.66 . * F 2.86 1.68 Glu 148 . . . . T T . 1.46 −0.73 . * F3.40 1.61 Asp 149 . . . . T T . 1.44 −0.74 . . F 3.06 2.19 Gly 150 . . .. . T C 1.14 −0.74 . . F 2.52 1.35 Pro 151 . . . . . . C 0.89 −0.76 . .F 1.98 1.04 Ser 152 . . . B . . C 0.89 −0.11 . * F 0.99 0.46 Met 153 . .B B . . . 0.19 0.29 * * . −0.30 0.75 Ser 154 . . B B . . . −0.12 0.64. * . −0.60 0.42 Val 155 . . B B . . . −0.08 0.70 . * . −0.60 0.45 Asn156 . . B B . . . −0.08 0.70 . * . −0.60 0.61 Phe 157 . . B B . . .−0.12 0.51 . * . −0.60 0.71 Thr 158 . . . B T . . −0.19 0.56 . * F −0.050.94 Ser 159 . . . . . T C −0.70 0.49 . * F 0.15 0.31 Pro 160 . . . . TT . 0.16 0.77 . * F 0.35 0.30 Gly 161 . . . . T T . 0.12 −0.01 . . F1.25 0.35 Cys 162 A . . . . T . −0.07 −0.00 . . . 0.70 0.35 Leu 163 A A. . . . . −0.36 0.30 * . . −0.30 0.16 Asp 164 A A . . . . . −0.01 0.49 *. . −0.60 0.16 His 165 A A . . . . . −0.04 0.06 * * . −0.30 0.60 Ile 166A A . . . . . 0.34 0.24 * . . −0.15 1.13 Met 167 A A . . . . . 1.06−0.44 * * . 0.45 1.36 Lys 168 A A . . . . . 1.91 −0.44 * * . 0.45 1.99Tyr 169 A A . . . . . 1.24 −0.94 * . F 0.90 5.69 Lys 170 A A . . . . .0.42 −1.06 * . F 0.90 3.08 Lys 171 A A . . . . . 1.36 −1.03 * . F 0.901.14 Lys 172 A A . . . . . 1.37 −1.03 * * F 0.90 1.46 Cys 173 . A B . .. . 0.98 −1.29 * . . 0.60 0.74 Val 174 . A B . . . . 0.92 −0.86 * . .0.60 0.36 Lys 175 . A B . . . . 0.07 −0.47 * . F 0.45 0.24 Ala 176 . A B. . . . −0.27 0.21 * . F 0.01 0.38 Gly 177 . . B . . T . −0.31 0.56 * .F 0.27 0.53 Ser 178 . . . . . T C 0.14 −0.09 . * F 1.53 0.44 Leu 179 . .. . T T . 1.00 0.34 * * F 1.29 0.68 Trp 180 . . . . T T . 0.07 0.24 * *F 1.60 1.11 Asp 181 . . . . . T C 0.34 0.50 . * F 0.79 0.58 Pro 182 . .. . T T . 0.10 0.60 . * F 0.98 1.01 Asn 183 . . . . T T . −0.27 0.41 * .F 0.67 0.97 Ile 184 A . . . . T . 0.59 0.07 * * . 0.26 0.31 Thr 185 A .. . . . . 0.92 0.07 * * . −0.10 0.40 Ala 186 A . . . . . . 0.92 −0.36. * . 0.50 0.50 Cys 187 A . . . . T . 1.13 −0.36 . . . 0.85 1.15 Lys 188A . . . . T . 1.13 −1.04 . . F 1.30 1.38 Lys 189 A . . . . T . 1.71−1.53 * . F 1.30 2.37 Asn 190 A . . . . T . 1.17 −1.54 * . F 1.30 6.38Glu 191 A . . . . . . 1.76 −1.47 * . F 1.10 2.37 Glu 192 A . . . . . .1.57 −1.47 . * F 1.10 2.05 Thr 193 A . . B . . . 1.52 −0.83 * * F 0.750.95 Val 194 A . . B . . . 0.78 −0.83 . * F 0.75 0.88 Glu 195 A . . B .. . 0.47 −0.04 . * . 0.30 0.44 Val 196 A . . B . . . 0.16 0.44 . * .−0.60 0.44 Asn 197 . . B B . . . −0.16 0.44 . * . −0.60 0.85 Phe 198 . .B B . . . −0.06 0.29 . * . −0.30 0.71 Thr 199 . . B B . . . −0.01 0.71. * F −0.30 1.48 Thr 200 . . B B . . . −0.36 0.76 . * F −0.33 0.76 Thr201 . . . . . T C 0.50 0.79 . * F 0.39 0.87 Pro 202 . . . . . T C 0.610.40 . . F 0.81 0.97 Leu 203 . . . . T T . 1.07 −0.09 * . F 1.88 1.32Gly 204 . . . . . T C 0.78 0.19 * . F 1.20 1.43 Asn 205 . . . . . T C0.50 0.31 * . F 0.93 0.91 Arg 206 . . B . . T . 0.00 0.39 * . . 0.611.12 Tyr 207 . . B . . T . −0.68 0.39 * * . 0.34 0.93 Met 208 . . B . .T . 0.13 0.64 * . . −0.08 0.41 Ala 209 . . B B . . . 0.44 0.64 * . .−0.60 0.36 Leu 210 . . B B . . . 0.14 1.14 * . . −0.60 0.31 Ile 211 . .B B . . . −0.28 0.77 * * . −0.60 0.42 Gln 212 . . B B . . . −0.92 0.64 .. . −0.60 0.60 His 213 . . B B . . . −1.21 0.83 . . . −0.60 0.51 Ser 214. . B B . . . −0.97 0.83 . . . −0.60 0.51 Thr 215 . . B B . . . −0.860.57 . . . −0.60 0.29 Ile 216 . . B B . . . −0.27 0.96 . . . −0.60 0.19Ile 217 . . B B . . . −0.27 0.84 . . . −0.60 0.19 Gly 218 . . B B . . .−1.09 0.86 * . . −0.60 0.22 Phe 219 . . B B . . . −1.49 1.01 * . . −0.600.24 Ser 220 . . . B . . C −1.18 1.11 * . . −0.40 0.29 Gln 221 . . B B .. . −0.50 0.43 * . . −0.60 0.51 Val 222 . . B B . . . 0.36 0.43 * . .−0.60 0.92 Phe 223 A . . B . . . 0.70 0.14 * . . −0.30 0.93 Glu 224 A .. B . . . 1.44 0.16 * . F −0.15 0.93 Pro 225 A A . . . . . 1.79 −0.24 *. F 0.60 2.51 His 226 A A . . . . . 1.79 −0.89 * . F 0.90 5.79 Gln 227 AA . . . . . 2.33 −1.27 * * F 0.90 5.79 Lys 228 A A . . . . . 3.14−0.79 * * F 0.90 5.40 Lys 229 A A . B . . . 2.56 −1.21 * * F 0.90 7.77Gln 230 A A . B . . . 2.47 −1.21 . * F 0.90 4.53 Thr 231 . A B B . . .1.64 −1.23 . * F 0.90 3.04 Arg 232 . A B B . . . 0.79 −0.59 * * F 0.901.13 Ala 233 . A B B . . . −0.14 0.06 * * F −0.15 0.48 Ser 234 . . B B .. . −0.40 0.34 . * . −0.30 0.23 Val 235 . . B B . . . −1.26 0.29 . * .−0.30 0.19 Val 236 . . B B . . . −1.26 0.93 . * . −0.60 0.14 Ile 237 . .B B . . . −1.71 0.91 * * . −0.60 0.15 Pro 238 . . B B . . . −1.12 0.96. * . −0.60 0.20 Val 239 . . B B . . . −1.12 0.31 . * . −0.30 0.44 Thr240 . . B B . . . −0.27 0.06 . * F 0.15 0.84 Gly 241 . . . B . . C 0.24−0.63 . * F 1.55 0.94 Asp 242 . . . . . T C 0.54 −0.63 . * F 2.40 1.26Ser 243 . . . . . T C 0.44 −0.77 . . F 2.55 0.88 Glu 244 . . . . . T C0.44 −0.77 . * F 3.00 1.28 Gly 245 . . B . . T . 0.76 −0.56 . * F 2.350.57 Ala 246 . . B B . . . 0.29 −0.16 . * F 1.35 0.74 Thr 247 . . B B .. . −0.02 0.14 . * . 0.30 0.35 Val 248 . . B B . . . 0.07 0.63 . . .−0.30 0.51 Gln 249 . . B B . . . −0.18 0.63 . * . −0.60 0.78 Leu 250 . .B B . . . −0.53 0.89 . * . −0.60 0.85 Thr 251 . . B B . . . −0.16 1.19. * . −0.60 0.99 Pro 252 . . B B . . . −0.16 0.97 . * F −0.45 0.89 Tyr253 . . . B T . . 0.03 1.06 * * F 0.10 1.55 Phe 254 . . B . . T . −0.310.94 * . . −0.20 0.58 Pro 255 . . . . T T . 0.20 0.89 * . F 0.35 0.37Thr 256 . . . . T T . 0.51 0.84 * . F 0.35 0.31 Cys 257 . . . . T T .0.06 0.09 * . F 0.65 0.61 Gly 258 . . . . T T . −0.59 −0.13 * * F 1.250.21 Ser 259 . . . . T T . 0.22 0.13 * . F 0.65 0.10 Asp 260 . . B . . T. 0.40 −0.36 * * F 0.85 0.37 Cys 261 . . B . . T . 0.76 −0.43 * * . 0.980.51 Ile 262 . . B . . . . 1.08 −0.86 * * . 1.36 0.77 Arg 263 . . B . .. . 1.11 −0.81 * * . 1.64 0.45 His 264 . . . . T T . 0.56 −0.33 * * .2.37 1.22 Lys 265 . . . . T T . −0.30 −0.26 * * F 2.80 1.30 Gly 266 . .. . T T . −0.44 −0.30 * * F 2.37 0.49 Thr 267 . . B . . T . −0.220.39 * * F 1.09 0.30 Val 268 . . B B . . . −0.54 0.46 . * . −0.04 0.08Val 269 . . B B . . . −0.51 0.89 . * . −0.32 0.12 Leu 270 . . B B . . .−0.87 0.86 . * . −0.60 0.15 Cys 271 . . B . . T . −0.87 0.86 . . . −0.200.29 Pro 272 . . B . . T . −1.41 0.64 . . F −0.05 0.39 Gln 273 . . . . TT . −0.77 0.64 . . F 0.35 0.35 Thr 274 . . . . T T . −0.61 0.39 . . F0.80 1.01 Gly 275 . . B . . . . −0.01 0.60 . * F −0.25 0.56 Val 276 . .B . . T . −0.16 0.60 . * . −0.20 0.50 Pro 277 . . B . . T . 0.06 0.89. * . −0.20 0.29 Phe 278 . . B . . T . 0.06 0.40 . * . 0.14 0.49 Pro 279. . B . . T . 0.37 0.37 . . . 0.93 1.05 Leu 280 . . B . . . . 0.76 0.13. . F 1.22 1.09 Asp 281 . . . . T T . 1.31 −0.30 . * F 2.76 2.52 Asn 282. . . . T T . 1.57 −0.70 . . F 3.40 2.19 Asn 283 . . . . T T . 2.06−1.13 . . F 3.06 5.31 Lys 284 . . . . T T . 1.92 −1.39 . . F 2.85 4.91Ser 285 . . . . . . C 2.39 −0.96 . . F 2.24 3.02 Lys 286 . . . . . T C2.10 −0.93 . . F 2.23 1.86 Pro 287 . . . . T T . 1.29 −0.41 . . F 1.770.98 Gly 288 . . . . T T . 1.08 0.27 . . F 1.30 0.60 Gly 289 . . . . T T. 0.22 0.31 . * F 1.17 0.47 Trp 290 . . B B . . . −0.29 1.00 * . . −0.210.25 Leu 291 . . B B . . . −1.14 1.26 . . . −0.34 0.21 Pro 292 . . B B .. . −1.74 1.51 . . . −0.47 0.17 Leu 293 . . B B . . . −1.70 1.77 . . .−0.60 0.14 Leu 294 . . B B . . . −2.17 1.24 . . . −0.60 0.22 Leu 295 . .B B . . . −2.69 1.24 . . . −0.60 0.12 Leu 296 . . B B . . . −2.73 1.50 .. . −0.60 0.12 Ser 297 . . B B . . . −3.11 1.46 . . . −0.60 0.11 Leu 298. . B B . . . −2.61 1.27 . . . −0.60 0.13 Leu 299 A . . B . . . −2.091.07 . . . −0.60 0.23 Val 300 A . . B . . . −2.13 1.30 . . . −0.60 0.18Ala 301 A . . B . . . −2.13 1.56 . . . −0.60 0.16 Thr 302 A . . B . . .−2.69 1.56 . . . −0.60 0.16 Trp 303 . . B B . . . −2.47 1.51 . . . −0.600.16 Val 304 . . B B . . . −2.00 1.37 . . . −0.60 0.16 Leu 305 . . B B .. . −2.03 1.30 . . . −0.60 0.11 Val 306 . . B B . . . −1.69 1.50 . . .−0.60 0.07 Ala 307 . . B B . . . −2.19 1.34 . . . −0.60 0.15 Gly 308 . .B B . . . −2.50 1.39 . . . −0.60 0.15 Ile 309 A . . B . . . −1.931.31 * * . −0.60 0.21 Tyr 310 A . . B . . . −1.01 1.59 * * . −0.60 0.21Leu 311 A . . B . . . −0.19 1.09 * * . −0.60 0.42 Met 312 A . . B . . .0.40 1.16 * * . −0.60 0.82 Trp 313 A . . B . . . 0.86 0.47 * * . −0.600.91 Arg 314 A . . B . . . 0.86 −0.29 . * . 0.45 2.16 His 315 A . . B .. . 1.14 −0.29 * . . 0.45 1.53 Glu 316 A . . B . . . 2.00 −0.90 * . .0.75 2.91 Arg 317 A A . . . . . 2.29 −1.81 * . F 0.90 2.97 Ile 318 A A .. . . . 2.28 −1.33 * . F 0.90 3.15 Lys 319 . A . . T . . 1.47 −1.44 * .F 1.30 2.43 Lys 320 . A . B T . . 1.20 −0.66 * * F 1.30 1.08 Thr 321 . A. B . . C 0.89 −0.27 * . F 0.80 2.06 Ser 322 . A . B . . C 0.47−0.47 * * F 0.80 1.48 Phe 323 . . B B . . . 1.04 0.01 * . F 0.00 1.07Ser 324 . . B B . . . 0.19 0.50 . . F −0.30 1.07 Thr 325 . . B B . . .−0.67 0.70 . . F −0.45 0.66 Thr 326 . . B B . . . −0.57 1.00 . . F −0.450.63 Thr 327 . . B B . . . −0.48 0.64 . . F −0.45 0.72 Leu 328 . . B B .. . −0.67 0.69 . * F −0.45 0.78 Leu 329 . . B B . . . −0.32 0.89 * * F−0.45 0.38 Pro 330 . . B B . . . −0.87 0.40 * . F −0.15 0.52 Pro 331 . .B B . . . −1.37 0.56 * * F −0.45 0.47 Ile 332 . . B B . . . −1.910.56 * * F −0.45 0.47 Lys 333 . . B B . . . −1.96 0.51 * * F −0.45 0.23Val 334 . . B B . . . −1.39 0.73 . . . −0.60 0.11 Leu 335 . . B B . . .−1.39 1.06 . * . −0.60 0.24 Val 336 . . B B . . . −1.48 0.80 * * . −0.600.19 Val 337 . . B B . . . −0.59 1.19 * * . −0.60 0.34 Tyr 338 . . B . .T . −1.52 0.54 * . . −0.20 0.71 Pro 339 A . . . . T . −1.33 0.54 . . F−0.05 0.67 Ser 340 A . . . T T . −1.22 0.47 . * F 0.35 0.48 Glu 341 A .. . . T . −0.40 0.61 . . F −0.05 0.27 Ile 342 A . . B . . . 0.42 0.36 .. . −0.30 0.24 Cys 343 A . . B . . . 0.36 0.43 . * . −0.60 0.24 Phe 344A . . B . . . −0.32 0.53 . * . −0.60 0.20 His 345 A . . B . . . −0.691.21 . * . −0.60 0.20 His 346 . . B B . . . −0.93 1.10 . * . −0.60 0.20Thr 347 . . . B T . . −0.74 1.29 . * . −0.20 0.36 Ile 348 . . . B T . .−0.39 1.29 * . . −0.20 0.23 Cys 349 . . . B T . . 0.31 1.27 * . . −0.200.24 Tyr 350 . . . B T . . −0.36 0.77 * . . −0.20 0.29 Phe 351 . . B B .. . −1.13 1.07 * . . −0.60 0.36 Thr 352 A . . B . . . −0.82 1.07 * . .−0.60 0.56 Glu 353 A . . B . . . 0.07 0.90 * . . −0.60 0.61 Phe 354 A .. B . . . 0.70 0.54 * . . −0.45 1.14 Leu 355 A . . B . . . 0.28 0.26 * *. −0.15 1.07 Gln 356 A . . B . . . 1.09 0.34 * * . −0.30 0.33 Asn 357 .. . B T . . 1.10 0.34 * * . 0.10 0.75 His 358 . . . B . . C 1.10−0.06 * * . 0.65 1.22 Cys 359 . . . . T T . 0.94 −0.74 . * . 1.55 1.22Arg 360 A . . . . T . 0.87 −0.50 * * F 1.15 0.56 Ser 361 A . . . . T .0.06 −0.21 . * F 0.85 0.29 Glu 362 A . . . . T . 0.06 −0.03 . * F 0.850.45 Val 363 A A . . . . . 0.13 −0.60 * * . 0.60 0.40 Ile 364 A A . . .. . 0.51 −0.60 * * . 0.60 0.59 Leu 365 A A . . . . . 0.40 −0.07 * * .0.30 0.36 Glu 366 A A . . . . . 0.74 0.33 * . . −0.30 0.84 Lys 367 A A .. . . . 0.79 −0.31 * . F 0.60 2.38 Trp 368 A A . . . . . 1.69 −1.00 . .F 0.90 5.78 Gln 369 A A . . . . . 1.69 −1.69 . . F 0.90 6.68 Lys 370 A A. . . . . 1.91 −1.00 * . F 0.90 2.34 Lys 371 A A . . . . . 1.91 −0.50 *. F 0.90 2.25 Lys 372 A A . . . . . 1.27 −1.41 * . F 0.90 2.25 Ile 373 AA . . . . . 1.21 −1.20 . . . 0.75 1.11 Ala 374 . A B . . . . 1.00 −0.77. . . 0.60 0.55 Glu 375 . A B . . . . 0.10 −0.34 . . . 0.30 0.43 Met 376. A B . . . . 0.06 0.30 * . . −0.30 0.45 Gly 377 . . B . . T . −0.280.01 * . . 0.10 0.77 Pro 378 A . . . . T . −0.20 0.43 . . . −0.20 0.47Val 379 A . . . . T . −0.20 1.11 . . . −0.20 0.39 Gln 380 A . . . . T .−0.51 1.00 . . . −0.20 0.40 Trp 381 A A . . . . . 0.09 1.06 . . . −0.600.37 Leu 382 A A . . . . . 0.48 1.03 . . . −0.60 0.87 Ala 383 A A . . .. . 0.73 0.39 . . . −0.15 1.00 Thr 384 A A . . . . . 1.00 −0.01 * . F0.60 1.91 Gln 385 A A . . . . . 0.41 −0.43 * . F 0.60 2.34 Lys 386 A A .. . . . 0.70 −0.61 * . F 0.90 2.34 Lys 387 A A . . . . . 1.56 −1.11 * .F 0.90 2.71 Ala 388 A A . . . . . 1.29 −1.60 * . F 0.90 3.12 Ala 389 A A. . . . . 0.74 −1.36 * . F 0.90 1.16 Asp 390 A A . . . . . 0.04 −0.71 *. F 0.75 0.43 Lys 391 A A . . . . . −0.81 0.07 * . . −0.30 0.37 Val 392. A B . . . . −1.67 0.26 * . . −0.30 0.30 Val 393 . A B . . . . −1.380.44 * . . −0.60 0.15 Phe 394 . A B . . . . −0.79 0.83 * . . −0.60 0.10Leu 395 . A B . . . . −0.79 1.23 * . . −0.60 0.22 Leu 396 . A B . . . .−1.69 0.59 * * . −0.60 0.49 Ser 397 . A . . T . . −0.83 0.59 * . F −0.050.42 Asn 398 . . . . T . . −0.28 0.20 * . F 0.45 0.81 Asp 399 . . . . TT . −0.43 −0.10 * . F 1.40 1.32 Val 400 . . . . T T . −0.29 −0.14 * . F1.25 0.73 Asn 401 . . B . T T . 0.52 0.04 * * F 0.65 0.24 Ser 402 . . B. . T . 0.48 −0.36 * . . 0.70 0.24 Val 403 . . B . . . . 0.17 0.07 * . .0.21 0.32 Cys 404 . . B . . T . −0.50 −0.09 * . . 1.32 0.29 Asp 405 . .B . . T . 0.01 0.09 . . F 1.18 0.12 Gly 406 . . . . T T . 0.06 0.13 . .F 1.89 0.16 Thr 407 . . . . T T . 0.06 −0.51 . . F 3.10 0.58 Cys 408 . .B . . T . 0.91 −0.70 . . F 2.39 0.47 Gly 409 . . . . T T . 1.23 −0.70 .. F 2.48 0.81 Lys 410 . . . . T T . 0.93 −0.70 . . F 2.17 0.56 Ser 411 .. . . . T C 1.07 −0.80 . . F 1.81 1.40 Glu 412 . . . . . . C 1.08 −0.94. . F 1.30 2.18 Gly 413 . . . . . . C 1.74 −0.99 . * F 1.64 1.46 Ser 414. . . . . T C 2.09 −0.99 . * F 2.18 1.89 Pro 415 . . . . . T C 1.74−0.97 . * F 2.52 1.75 Ser 416 . . . . . T C 2.04 −0.59 . . F 2.86 2.38Glu 417 . . . . T T . 2.04 −0.61 . . F 3.40 3.07 Asn 418 . . . . T . .2.09 −1.00 . . F 2.86 3.32 Ser 419 . . . . T T . 2.09 −1.04 . . F 2.933.32 Gln 420 . . . . T T . 2.09 −1.04 . . F 2.80 2.57 Asp 421 . . . . TT . 1.72 −0.61 . . F 2.67 2.47 Ser 422 . . . . T T . 0.91 −0.44 . . F2.09 0.99 Ser 423 . . . . . T C 0.52 −0.14 . . F 2.10 0.47 Pro 424 . . B. . T . 0.43 −0.11 . . . 1.54 0.36 Cys 425 . . B . . T . 0.04 0.31 . . .0.73 0.34 Leu 426 . . B . . T . −0.34 0.36 . . . 0.52 0.33

Among highly preferred fragments in this regard are those that compriseregions of IL17RLP that combine several structural features, such asseveral features set out above.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, the cDNAclone contained in ATCC™ Deposit No. 209198. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,more preferably at least about 25 nt, still more preferably at leastabout 30 nt, and even more preferably about 30-70 (e.g., 30, 35, 40, 45,50, 55, 60, 65, and/or 70 (of course, fragment lengths in addition tothose recited herein are also useful)) nt of the referencepolynucleotide. These are useful as diagnostic probes and primers asdiscussed above and in more detail below.

By a portion of a polynucleotide of “at least 20 nt in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in FIGS. 1A, 1B, and 1C (SEQ IDNO:1)). Of course, a polynucleotide which hybridizes only to a poly Asequence (such as the 3′ terminal poly(A) tract of the IL17RLP cDNAshown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1)), or to a complementarystretch of T (or U) residues, would not be included in a polynucleotideof the invention used to hybridize to a portion of a nucleic acid of theinvention, since such a polynucleotide would hybridize to any nucleicacid molecule containing a poly (A) stretch or the complement thereof(e.g., practically any double-stranded cDNA clone).

In preferred embodiments, polynucleotides which hybridize to thereference polynucleotides disclosed herein encode polypeptides whicheither retain substantially the same biological function or activity asthe mature form of the IL17RLP polypeptide encoded by the polynucleotidesequence depicted in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) or the clonecontained in the deposit (HAPOR40).

Alternative embodiments are directed to polynucleotides which hybridizeto the reference polynucleotide (i.e., a polynucleotide sequencedisclosed herein), but do not retain biological activity. While thesepolynucleotides do not retain biological activity, they have uses, suchas, for example, as probes for the polynucleotides of SEQ ID NO:1, forrecovery of the polynucleotides, as diagnostic probes, and as PCRprimers.

As indicated, nucleic acid molecules of the present invention whichencode a IL17RLP polypeptide may include, but are not limited to thoseencoding the amino acid sequence of the mature polypeptide, by itself;and the coding sequence for the mature polypeptide and additionalsequences, such as those encoding the about 19 amino acid leader orsecretory sequence, such as a pre-, or pro- or prepro-protein sequence;the coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences.

Also encoded by nucleic acids of the invention are the above proteinsequences together with additional, non-coding sequences, including forexample, but not limited to introns and non-coding 5′ and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylationsignals, for example—ribosome binding and stability of mRNA; anadditional coding sequence which codes for additional amino acids, suchas those which provide additional functionalities.

Thus, the sequence encoding the polypeptide may be fused to a markersequence, such as a sequence encoding a peptide which facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described by Gentzand colleagues (Proc. Natl. Acad. Sci. USA 86:821-824 (1989)), forinstance, hexa-histidine provides for convenient purification of thefusion protein. The “HA” tag is another peptide useful for purificationwhich corresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson and coworkers (Cell 37:767(1984)). As discussed below, other such fusion proteins include theIL17RLP fused to Fc at the N- or C-terminus.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the IL17RLP protein. Variants may occur naturally, suchas a natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985)). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the IL17RLP protein or portions thereof.Also especially preferred in this regard are conservative substitutions.

Most highly preferred are nucleic acid molecules encoding the matureprotein having the amino acid sequence shown in SEQ ID NO:2, SEQ IDNO:18, or the mature IL17RLP amino acid sequence encoded by thedeposited cDNA clone.

Most highly preferred are nucleic acid molecules encoding theextracellular domain of the protein having the amino acid sequence shownin SEQ ID NO:2, SEQ ID NO:18, or the extracellular domain of the IL17RLPamino acid sequence encoded by the deposited cDNA clone.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding the IL17RLP polypeptide having the complete amino acid sequencein SEQ ID NO:2 (i.e., positions −19 to 407 of SEQ ID NO:2); (b) anucleotide sequence encoding the IL17RLP polypeptide having the completeamino acid sequence in SEQ ID NO:2 excepting the N-terminal methionine(i.e., positions −18 to 407 of SEQ ID NO:2); (c) a nucleotide sequenceencoding the predicted mature IL17RLP polypeptide having the amino acidsequence at positions 1 to 407 in SEQ ID NO:2; (d) a nucleotide sequenceencoding a polypeptide comprising the predicted extracellular domain ofthe IL17RLP polypeptide having the amino acid sequence at positions 1 to271 in SEQ ID NO:2; (e) a nucleotide sequence encoding a soluble IL17RLPpolypeptide having the predicted extracellular and intracellulardomains, but lacking the predicted transmembrane domain; (f) anucleotide sequence encoding the IL17RLP polypeptide having the completeamino acid sequence encoded by the human cDNA contained in ATCC™ DepositNo. 209198; (g) a nucleotide sequence encoding the IL17RLP polypeptidehaving the complete amino acid sequence excepting the N-terminalmethionine encoded by the human cDNA contained in ATCC™ Deposit No.209198; (h) a nucleotide sequence encoding the mature IL17RLPpolypeptide having the amino acid sequence encoded by the human cDNAcontained in ATCC™ Deposit No. 209198; (i) a nucleotide sequenceencoding the extracellular domain of the IL17RLP polypeptide having theamino acid sequence encoded by the human cDNA contained in ATCC™ DepositNo. 209198; and (j) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c), (d), (e), (0, (g), (h) or (i)above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 80%, 85%, 90% identical, and more preferably at least 92%, 95%,96%, 97%, 98%, 99% or 100% identical, to any of the nucleotide sequencesin (a), (b), (c), (d), (e), (f), (g), (h) or (i), above, or apolynucleotide which hybridizes under stringent hybridization conditionsto a polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h) or (i),above. This polynucleotide which hybridizes does not hybridize understringent hybridization conditions to a polynucleotide having anucleotide sequence consisting of only A residues or of only T residues.An additional nucleic acid embodiment of the invention relates to anisolated nucleic acid molecule comprising a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion of a IL17RLPpolypeptide having an amino acid sequence in (a), (b), (c), (d), (e),(f), (g) or (h), above. A further nucleic acid embodiment of theinvention relates to an isolated nucleic acid molecule comprising apolynucleotide which encodes the amino acid sequence of a IL17RLPpolypeptide having an amino acid sequence which contains at least oneconservative amino acid substitution, but not more than 50 conservativeamino acid substitutions, even more preferably, not more than 40conservative amino acid substitutions, still more preferably not morethan 30 conservative amino acid substitutions, and still even morepreferably not more than 20 conservative amino acid substitutions. Ofcourse, in order of ever-increasing preference, it is highly preferablefor a polynucleotide which encodes the amino acid sequence of a IL17RLPpolypeptide to have an amino acid sequence which contains not more than10-20, 10-15, 7-15, 7-10, 5-10, 3-7, 3-5, 2-5, 1-5, 1-3, 10, 9, 8, 7, 6,5, 4, 3, 2 or 1 conservative amino acid substitutions.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofIL17RLP polypeptides or peptides by recombinant techniques.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a IL17RLPpolypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding theIL17RLP polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100° A identicalto, for instance, the nucleotide sequence shown in FIGS. 1A, 1B, and 1Cor to the nucleotides sequence of the deposited cDNA clone can bedetermined conventionally using known computer programs such as theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). Bestfit uses the local homology algorithmof Smith and Waterman to find the best segment of homology between twosequences (Advances in Applied Mathematics 2:482-489 (1981)). When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference nucleotide sequence and that gaps in homology ofup to 5% of the total number of nucleotides in the reference sequenceare allowed. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag and colleagues (Comp. App. Biosci. 6:237-245 (1990)). In asequence alignment the query and subject sequences are both DNAsequences. An RNA sequence can be compared by converting U's to T's. Theresult of said global sequence alignment is in percent identity.Preferred parameters used in a FASTDB alignment of DNA sequences tocalculate percent identity are: Matrix=Unitary, k-tuple=4, MismatchPenalty=1, Joining Penalty=30, Randomization Group Length=0, CutoffScore=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or thelength of the subject nucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

In certain preferred embodiments, IL17RLP proteins of the inventioncomprise fusion proteins as described herein wherein the IL17RLPpolypeptides are those described as n¹-m¹, n²-m², and/or n³-m³ herein.In preferred embodiments, the application is directed to nucleic acidmolecules at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the nucleic acid sequences encoding polypeptides having theamino acid sequence of the specific N- and C-terminal deletions recitedherein. Polynucleotides encoding these polypeptides are also encompassedby the invention.

The present application is directed to nucleic acid molecules at least80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to thenucleic acid sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) or tothe nucleic acid sequence of the deposited cDNA, irrespective of whetherthey encode a polypeptide having IL17RLP activity. This is because evenwhere a particular nucleic acid molecule does not encode a polypeptidehaving IL17RLP activity, one of skill in the art would still know how touse the nucleic acid molecule, for instance, as a hybridization probe ora polymerase chain reaction (PCR) primer. Uses of the nucleic acidmolecules of the present invention that do not encode a polypeptidehaving IL17RLP activity include, inter alia, (1) isolating the IL17RLPgene or allelic variants thereof in a cDNA library; (2) in situhybridization (e.g., “FISH”) to metaphase chromosomal spreads to provideprecise chromosomal location of the IL17RLP gene, as described by Vermaand colleagues (Human Chromosomes: A Manual of Basic Techniques,Pergamon Press, New York (1988)); and Northern Blot analysis fordetecting IL17RLP mRNA expression in specific tissues.

Preferred, however, are nucleic acid molecules comprising, oralternatively consisting of, sequences at least 80%, 85%, 90%, 92%, 95%,96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence shownin FIGS. 1A, 1B, and 1C (SEQ ID NO:1) or to the nucleic acid sequence ofthe deposited cDNA which do, in fact, encode a polypeptide havingIL17RLP protein activity. By “a polypeptide having IL17RLP activity” isintended polypeptides exhibiting activity similar, but not necessarilyidentical, to an activity of the mature or soluble form of the IL17RLPprotein of the invention, as measured in a particular biological assay.For example, the IL17RLP protein of the present invention modulates IL-6secretion from NIH-3T3 cells. An in vitro ELISA assay which quantitatesthe amount of IL-6 secreted from cells in response to treatment withcytokines or the soluble extracellular domains of cytokine receptors hasbeen described (Yao, Z., et al., Immunity 3:811-821 (1995)). Briefly,the assay involves plating the target cells at a density ofapproximately 5×10⁶ cells/mL in a volume of 500 microliters in the wellsof a 24 well flat-bottomed culture plate (Costar). The cultures are thentreated with various concentrations of the cytokine or the solubleextracellular domain of cytokine receptor in question The cells are thencultured for 24 hours at 37° C. At this time, 50 microliters ofsupernatant is removed and assayed for the quantity of IL-6 essentiallyas described by the manufacturer (Genzyme, Boston, Mass.). IL-6 levelsare then calculated by reference to a standard curve constructed withrecombinant IL-17 cytokine. Such activity is useful for determining thelevel of IL17RLP-mediated IL-6 secretion.

IL17RLP protein modulates immune system cell proliferation anddifferentiation in a dose-dependent manner in the above-described assay.Thus, “a polypeptide having IL17RLP protein activity” includespolypeptides that also exhibit any of the same stimulatory activities inthe above-described assays in a dose-dependent manner. Although thedegree of dose-dependent activity need not be identical to that of theIL17RLP protein, preferably, “a polypeptide having IL17RLP proteinactivity” will exhibit substantially similar dose-dependence in a givenactivity as compared to the IL17RLP protein (i.e., the candidatepolypeptide will exhibit greater activity or not more than about 25-foldless and, preferably, not more than about tenfold less activity relativeto the reference IL17RLP protein).

Lymphocyte proliferation is another in vitro assay which may beperformed to determine the activity of IL17RLP, soluble, extracellulardomains of IL17RLP, and agonists and antagonists (e.g., anti-IL17RLPantibodies). For example, Yao and colleagues (Immunity 3:811-821 (1995))have recently described an in vitro assay for determining the effects ofvarious cytokines and soluble cytokine receptors on the proliferation ofmurine leukocytes. Briefly, lymphoid organs are harvested aseptically,lymphocytes are isolated from the harvested organs, and the resultingcollection of lymphoid cells are suspended in standard culture medium asdescribed by Fanslow and coworkers (J. Immunol. 147:535-5540 (1991)).The lymphoid cell suspensions may then be divided into several differentsubclasses of lymphoid cells including splenic T-cells, lymph nodeB-cells, CD4⁺ and CD8⁺ T-cells, and mature adult thymocytes. For splenicT-cells, spleen cell suspensions (200×10⁶ cells) are incubated with CD11b mAb and class II MHC mAb for 30 min at 4° C., loaded on a T-cellpurification column (Pierce, Rockford, Ill.), and the T-cells elutedaccording to the manufacturer's instructions. Using this method, purityof the resulting T-cell populations should be >95% CD3⁺ and <1% sIgM⁺.For purification of lymph node subsets, B-cells are removed from byadherence to tissue culture dishes previously coated with goatanti-mouse IgG (10 μg/mL). Remaining cells were then incubated withanti-CD4 or anti-CD8 for 30 min at 4° C. then washed and placed ontissue culture dishes previously coated with goat anti-rat IgG (20micrograms per milliliter). After 45 min, nonadherent cells are removedand tested for purity by flow cytometry. CD4 and surface Ig-depletedcells should be >90% TCR-ab, CD8⁺, whereas CD8 and surface Ig-depletedcells should be >95% TCR-ab, CD4⁺. Finally, to enrich for mature adultthymocytes, cells are suspended at 10⁸/mL in 10% anti-HSA and 10% lowtox rabbit complement (Cedarlane, Ontario, Canada), incubated for 45 minat 37° C., and remaining viable cells isolated over FICOLL™-Hypaque(PHARMACIA™, Piscataway, N.J.). This procedure should yield between 90and 95% CD3^(hi) cells that are either CD4⁺8⁻ or CD4⁻8⁺.

To analyze the proliferative response of the above-described primarycell cultures, in vitro proliferation assays are set up in round bottomor flat bottom 96-well plates using 0.5−1.5×10⁵ cells/well. Forstimulation, T-cells are incubated with suboptimal concentrations(0.25-0.5 micrograms per milliliter) of Con A (SIGMA™, St. Louis, Mo.),PHA (0.25-0.5%; Difco, Detroit, Mich.), immobilized anti-CD3, orimmobilized anti-TCR-ab. Anti-CD3 and anti-TCR-ab are immobilized for >2hours at 37° C. before the addition of effector cells. Incubations aredone in the presence and absence of fixed CV-1/EBNA cells transfectedwith IL17RLP, muteins thereof, a control vector, or a control antigensuch as rCD40L (Armitage, et al., Nature 357:80 (1992)); Spriggs, etal., J. Exp. Med. 176:1543 (1992)). Surface expression of CD40L ismonitored by flow cytometry using a human CD40-Fc fusion protein. Cellcultures are pulsed overnight with [³H]-thymidine (1 microcurie perwell) for the last 18 hours of a 3 day culture. Labeled cultures arethen harvested on a 96-well Inotech harvester and radioactive countsdetected using a scintillation counter.

Like other cytokine receptors, IL17RLP exhibits activity on leukocytesincluding for example monocytes, lymphocytes and neutrophils. For thisreason IL17RLP is active in directing the proliferation anddifferentiation of these cell types. Such activity is useful for immuneenhancement or suppression, myeloprotection, stem cell mobilization,acute and chronic inflammatory control and treatment of leukemia. Assaysfor measuring such activity are well known in the art (Peters, et al.,Immun. Today 17:273 (1996); Young, et al., J. Exp. Med. 182:1111 (1995);Caux, et al., Nature 390:258 (1992); and Santiago-Schwarz, et al., Adv.Exp. Med. Biol. 378:7 (1995).

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 80%, 85%, 90%, 92%,95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequenceof the deposited cDNA or the nucleic acid sequence shown in FIGS. 1A,1B, and 1C (SEQ ID NO:1) will encode a polypeptide “having IL17RLPprotein activity.” In fact, since degenerate variants of thesenucleotide sequences all encode the same polypeptide, this will be clearto the skilled artisan even without performing the above describedcomparison assay. It will be further recognized in the art that, forsuch nucleic acid molecules that are not degenerate variants, areasonable number will also encode a polypeptide having IL17RLP proteinactivity. This is because the skilled artisan is fully aware of aminoacid substitutions that are either less likely or not likely tosignificantly effect protein function (e.g., replacing one aliphaticamino acid with a second aliphatic amino acid), as further describedbelow.

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in IL17RLP,or the complementary strand thereof, and/or to nucleotide sequencescontained in the deposited clone HAPOR40. In one embodiment, antisensesequence is generated internally by the organism, in another embodiment,the antisense sequence is separately administered (see, for example,O'Connor, J., Neurochem. 56:560 (1991), and Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Antisense technology can be used to control gene expressionthrough antisense DNA or RNA, or through triple-helix formation.Antisense techniques are discussed for example, in Okano, J., Neurochem.56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

In one embodiment, the IL17RLP antisense nucleic acid of the inventionis produced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the IL17RLP antisense nucleic acid. Such avector can remain episomal or become chromosomally integrated, as longas it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others know inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding IL17RLP, or fragments thereof, canbe by any promoter known in the art to act in vertebrate, preferablyhuman cells. Such promoters can be inducible or constitutive. Suchpromoters include, but are not limited to, the SV40 early promoterregion (Bernoist and Chambon, Nature 29:304-310 (1981), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al., Cell 22:787-797 (1980), the herpes thymidine promoter (Wagner etal., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatorysequences of the metallothionein gene (Brinster, et al., Nature296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of aN IL17RLPgene. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA,”referred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded IL17RLP antisense nucleic acids, a single strandof the duplex DNA may thus be tested, or triplex formation may beassayed. The ability to hybridize will depend on both the degree ofcomplementarity and the length of the antisense nucleic acid. Generally,the larger the hybridizing nucleic acid, the more base mismatches with aIL17RLP RNA it may contain and still form a stable duplex (or triplex asthe case may be). One skilled in the art can ascertain a tolerabledegree of mismatch by use of standard procedures to determine themelting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333-335. Thus, oligonucleotides complementary to either the 5′- or3′-non-translated, non-coding regions of the IL17RLP shown in FIGS. 1A,1B, and 1C could be used in an antisense approach to inhibit translationof endogenous IL17RLP mRNA. Oligonucleotides complementary to the 5′untranslated region of the mRNA should include the complement of the AUGstart codon. Antisense oligonucleotides complementary to mRNA codingregions are less efficient inhibitors of translation but could be usedin accordance with the invention. Whether designed to hybridize to the5′-, 3′- or coding region of IL17RLP mRNA, antisense nucleic acidsshould be at least six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Inspecific aspects the oligonucleotide is at least 10 nucleotides, atleast 17 nucleotides, at least 25 nucleotides or at least 50nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A.86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. 84:648-652(1987); PCT Publication No. WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., BioTechniques 6:958-976 (1988)) or intercalatingagents. (See, e.g., Zon, Pharm. Res. 5:539-549 (1988)). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res. 15:6625-6641 (1987)). The oligonucleotide is a2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (Nucl. Acids Res. 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A.85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to the IL17RLP coding regionsequence could be used, those complementary to the transcribeduntranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy IL17RLP mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA have thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, Nature 334:585-591 (1988).There are numerous potential hammerhead ribozyme cleavage sites withinthe nucleotide sequence of IL17RLP (FIGS. 1A, 1B, and 1C (SEQ ID NO:1)).Preferably, the ribozyme is engineered so that the cleavage recognitionsite is located near the 5′ end of the IL17RLP mRNA; i.e., to increaseefficiency and minimize the intracellular accumulation of non-functionalmRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express IL17RLPin vivo. DNA constructs encoding the ribozyme may be introduced into thecell in the same manner as described above for the introduction ofantisense encoding DNA. A preferred method of delivery involves using aDNA construct “encoding” the ribozyme under the control of a strongconstitutive promoter, such as, for example, pol III or pol II promoter,so that transfected cells will produce sufficient quantities of theribozyme to destroy endogenous IL17RLP messages and inhibit translation.Since ribozymes unlike antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

Endogenous gene expression can also be reduced by inactivating or“knocking out” the IL17RLP gene and/or its promoter using targetedhomologous recombination. (E.g., see Smithies et al., Nature 317:230-234(1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart. The contents of each of the documents recited in this paragraph isherein incorporated by reference in its entirety.

In other embodiments, antagonists according to the present inventioninclude soluble forms of IL17RLP (e.g., fragments of the IL17RLP shownin FIGS. 1A, 1B, and 1C (SEQ ID NO:2) that include the ligand bindingdomain from the extracellular region of the full length receptor). Suchsoluble forms of the IL17RLP, which may be naturally occurring orsynthetic, antagonize IL17RLP-mediated signaling by competing with thecell surface bound forms of the receptor for binding to IL-20 orIL-20-like ligands. Antagonists of the present invention also includeantibodies specific for IL17RLP ligands and IL17RLP-Fc fusion proteins.

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, and the production of IL17RLPpolypeptides or fragments thereof by recombinant or synthetictechniques. The vector may be, for example, a phage, plasmid, viral orretroviral vector. Retroviral vectors may be replication competent orreplication defective. In the latter case, viral propagation generallywill occur only in complementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe transcripts expressed by the constructs will preferably include atranslation initiating codon at the beginning and a termination codon(UAA, UGA or UAG) appropriately positioned at the end of the polypeptideto be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC™Accession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, 293 and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

Vectors preferred for use in bacteria include pHE4-5 (ATCC™ AccessionNo. 209311; and variations thereof), pQE70, pQE60 and pQE-9 (QIAGEN,Inc., supra); pBS vectors, Phagescript vectors, Bluescript vectors,pNH8A, pNH16a, pNH18A, pNH46A (STRATAGENE™); and ptrc99a, pKK223-3,pKK233-3, pDR540, pRIT5 (PHARMACIA™). Preferred expression vectors foruse in yeast systems include, but are not limited to, pYES2, pYD1,pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2,pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogen, Carlsbad,Calif.). Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44,pXT1, and pSG (STRATAGENE™); and pSVK3, pBPV, pMSG and pSVL(PHARMACIA™). Other suitable vectors will be readily apparent to theskilled artisan.

In one embodiment, the yeast Pichia pastoris is used to express IL17RLPprotein in a eukaryotic system. Pichia pastoris is a methylotrophicyeast which can metabolize methanol as its sole carbon source. A mainstep in the methanol metabolization pathway is the oxidation of methanolto formaldehyde using O₂. This reaction is catalyzed by the enzymealcohol oxidase. In order to metabolize methanol as its sole carbonsource, Pichia pastoris must generate high levels of alcohol oxidasedue, in part, to the relatively low affinity of alcohol oxidase for O₂.Consequently, in a growth medium depending on methanol as a main carbonsource, the promoter region of one of the two alcohol oxidase genes(AOX1) is highly active. In the presence of methanol, alcohol oxidaseproduced from the AOX1 gene comprises up to approximately 30% of thetotal soluble protein in Pichia pastoris. See, Ellis, S. B., et al.,Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77(1989); Tschopp, J. F., et al, Nucl. Acids Res. 15:3859-76 (1987). Thus,a heterologous coding sequence, such as, for example, an IL17RLPpolynucleotide of the present invention, under the transcriptionalregulation of all or part of the AOX1 regulatory sequence is expressedat exceptionally high levels in Pichia yeast grown in the presence ofmethanol.

In one example, the plasmid vector pPIC9K is used to express DNAencoding an IL17RLP polypeptide of the invention, as set forth herein,in a Pichea yeast system essentially as described in “Pichia Protocols:Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. TheHumana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of an IL17RLP protein of the invention byvirtue of the strong AOX1 promoter linked to the Pichia pastorisalkaline phosphatase (PHO) secretory signal peptide (i.e., leader)located upstream of a multiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as,pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG as required.

In one embodiment, high-level expression of a heterologous codingsequence, such as, for example, an IL17RLP polynucleotide of the presentinvention, may be achieved by cloning the heterologous polynucleotide ofthe invention into an expression vector such as, for example, pGAPZ orpGAPZalpha, and growing the yeast culture in the absence of methanol.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals (for example, Davis, et al., Basic Methods InMolecular Biology (1986)).

The polypeptide (e.g., the mature or the extracellular domain of IL17RLPof the invention) may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional or non-functional regions. For instance, aregion of additional amino acids, particularly charged amino acids, maybe added to the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification, or during subsequenthandling and storage. Also, peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to polypeptides to engender secretion or excretion, to improvestability and to facilitate purification, among others, are familiar androutine techniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to stabilize andpurify proteins. For example, EP-A-0 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobulin molecules together with another humanprotein or part thereof. In many cases, the Fc part in a fusion proteinis thoroughly advantageous for use in therapy and diagnosis and thusresults, for example, in improved pharmacokinetic properties (EP-A 0232262). On the other hand, for some uses it would be desirable to be ableto delete the Fc part after the fusion protein has been expressed,detected and purified in the advantageous manner described. This is thecase when Fc portion proves to be a hindrance to use in therapy anddiagnosis, for example when the fusion protein is to be used as antigenfor immunizations. In drug discovery, for example, human proteins, suchas hlL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5(Bennett, D., et al., J. Molecular Recognition 8:52-58 (1995); Johanson,K., et al., J. Biol. Chem. 270:9459-9471 (1995)).

The IL17RLP protein can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Polypeptides of the presentinvention include: products purified from natural sources, includingbodily fluids, tissues and cells, whether directly isolated or cultured;products of chemical synthetic procedures; and products produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, bacterial, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes. Thus, it is well known in the artthat the N-terminal methionine encoded by the translation initiationcodon generally is removed with high efficiency from any protein aftertranslation in all eukaryotic cells. While the N-terminal methionine onmost proteins also is efficiently removed in most prokaryotes, for someproteins this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., IL17RLP coding sequence), and/or to includegenetic material (e.g., heterologous polynucleotide sequences) that isoperably associated with IL17RLP polynucleotides of the invention, andwhich activates, alters, and/or amplifies endogenous IL17RLPpolynucleotides. For example, techniques known in the art may be used tooperably associate heterologous control regions (e.g., promoter and/orenhancer) and endogenous IL17RLP polynucleotide sequences via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication Number WO 96/29411, published Sep. 26, 1996;International Publication Number WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); andZijlstra et al., Nature 342:435-438 (1989), the disclosures of each ofwhich are incorporated by reference in their entireties).

Polypeptides and Fragments

The invention further provides an isolated IL17RLP polypeptidecomprising, or alternatively consisting of, the amino acid sequenceencoded by the deposited cDNA, or the amino acid sequence in SEQ IDNO:2, or a peptide or polypeptide comprising, or alternativelyconsisting of, a portion of the above polypeptides.

To improve or alter the characteristics of IL17RLP polypeptides, proteinengineering may be employed. Recombinant DNA technology known to thoseskilled in the art can be used to create novel mutant proteins ormuteins including single or multiple amino acid substitutions,deletions, additions or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions.

For instance, for many proteins, including the extracellular domain of amembrane associated protein or the mature form(s) of a secreted protein,it is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. For instance, Ron and colleagues (J. Biol. Chem.,268:2984-2988 (1993)) reported modified KGF proteins that had heparinbinding activity even if 3, 8, or 27 N-terminal amino acid residues weremissing. In the present case, since the protein of the invention is amember of the interleukin (IL)-17 receptor polypeptide family, deletionsof N-terminal amino acids up to the cysteine at position 5 of SEQ IDNO:2 may retain some biological activity such as ligand binding ormodulation of target cell activities. Polypeptides having furtherN-terminal deletions including the cysteine residue at position 5 in SEQID NO:2 would not be expected to retain such biological activitiesbecause it is known that this residue in the murine IL-17 receptorpolypeptide is likely required for forming a disulfide bridge to providestructural stability which is needed for ligand binding and theinitiation of the appropriate signal transduction pathways.

However, even if deletion of one or more amino acids from the N-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete, mature or extracellulardomain of the protein generally will be retained when less than themajority of the residues of the complete, mature or extracellular domainof the protein are removed from the N-terminus. Whether a particularpolypeptide lacking N-terminal residues of a complete protein retainssuch immunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence of the IL17RLP shown in SEQ ID NO:2, up to the cysteine residueat position number 5, and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising, oralternatively consisting of, the amino acid sequence of residues n¹-407of SEQ ID NO:2, where n¹ is an integer in the range of −19 to 5, and 5is the position of the first residue from the N-terminus of the completeIL17RLP polypeptide (shown in SEQ ID NO:2) believed to be required forligand binding activity of the IL17RLP protein. More in particular, theinvention provides polynucleotides encoding polypeptides comprising, oralternatively consisting of, a member of the group consisting of theamino acid sequence of residues of −18-407, −17-407, −16-407, −15-407,−14-407, −13-407, −12-407, −11-407, −10-407, −9-407, −8-407, −7-407,−6-407, −5-407, −4-407, −3-407, −2-407, −1-407, 1-407, 2-407, 3-407,4-407, and 5-407 of SEQ ID NO:2. Polypeptides encoded by thesepolynucleotides are also encompassed by the invention. The presentinvention is also directed to nucleic acid molecules comprising, oralternatively, consisting of, a polynucleotide sequence at least 80%,85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to thepolynucleotide sequence encoding the IL17RLP polypeptides describedabove. The present invention also encompasses the above polynucleotidesequences fused to a heterologous polynucleotide sequence. Polypeptidesencoded by these nucleic acids and/or polynucleotide sequences are alsoencompassed by the invention, as are polypeptides comprising, oralternatively consisting of, an amino acid sequence at least 80%, 85%,90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence described above, and polynucleotides that encode suchpolypeptides.

Similarly, many examples of biologically functional C-terminal deletionmuteins are known. For instance, Interferon gamma shows up to ten timeshigher activities by deleting 8-10 amino acid residues from the carboxyterminus of the protein (Dobeli, et al, J. Biotechnology 7:199-216(1988)). In the present case, since the protein of the invention is amember of the interleukin (IL)-17 receptor polypeptide family, deletionsof C-terminal amino acids up to the cysteine at position 340 of SEQ IDNO:2 may retain some biological activity such as ligand-binding.Polypeptides having further C-terminal deletions including the cysteineresidue at position 340 of SEQ ID NO:2 would not be expected to retainsuch biological activities because it is known that this residue in themurine IL-17 receptor polypeptide is likely required for forming adisulfide bridge to provide structural stability which is needed forreceptor binding and signal transduction.

However, even if deletion of one or more amino acids from the C-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete, mature or extracellulardomain of the protein generally will be retained when less than themajority of the residues of complete, mature or extracellular domain ofthe protein are removed from the C-terminus. Whether a particularpolypeptide lacking C-terminal residues of a complete protein retainssuch immunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art.

Accordingly, the present invention further provides polypeptides havingone or more residues from the carboxy terminus of the amino acidsequence of the IL17RLP shown in SEQ ID NO:2, up to the cysteine residueat position 340 of SEQ ID NO:2, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising, or alternatively consisting of, the amino acid sequence ofresidues-19-m¹ of the amino acid sequence in SEQ ID NO:2, where m¹ isany integer in the range of 340 to 407, and residue 340 is the positionof the first residue from the C-terminus of the complete IL17RLPpolypeptide (shown in SEQ ID NO:2) believed to be required for theIL17RLP protein to transfer its extracellular signal to the interior ofthe cell. More in particular, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, amember selected from the group consisting of the amino acid sequence ofresidues −19-340, −19-341, −19-342, −19-343, −19-344, −19-345, −19-346,−19-347, −19-348, −19-349, −19-350, −19-351, −19-352, −19-353, −19-354,−19-355, −19-356, −19-357, −19-358, −19-359, −19-360, −19-361, −19-362,−19-363, −19-364, −19-365, −19-366, −19-367, −19-368, −19-369, −19-370,−19-371, −19-372, −19-373, −19-374, =19-375, −19-376, −19-377, −19-378,−19-379, −19-380, −19-381, −19-382, −19-383, −19-384, −19-385, −19-386,−19-387, −19-388, −19-389, −19-390, −19-391, −19-392, −19-393, −19-394,−19-395, −19-396, −19-397, −19-398, −19-399, −19-400, −19-401, −19-402,−19-403, −19-404, −19-405, −19-406, and −19-407 of SEQ ID NO:2.Polypeptides encoded by these polynucleotides are also encompassed bythe invention. The present invention is also directed to nucleic acidmolecules comprising, or alternatively, consisting of, a polynucleotidesequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100%identical to the polynucleotide sequence encoding the IL17RLPpolypeptides described above. The present invention also encompasses theabove polynucleotide sequences fused to a heterologous polynucleotidesequence. Polypeptides encoded by these nucleic acids and/orpolynucleotide sequences are also encompassed by the invention, as arepolypeptides comprising, or alternatively consisting of, an amino acidsequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence described above, andpolynucleotides that encode such polypeptides.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini, which may bedescribed generally as comprising, or alternatively consisting of,residues n¹-m¹ of SEQ ID NO:2, where n¹ and m¹ are integers as describedabove.

Also included are a nucleotide sequence encoding a polypeptidecomprising, or alternatively consisting of, a portion of the completeIL17RLP amino acid sequence encoded by the cDNA clone contained in ATCC™Deposit No. 209198, where this portion excludes from 1 to about 23 aminoacids from the amino terminus of the complete amino acid sequenceencoded by the cDNA clone contained in ATCC™ Deposit No. 209198, or from1 to about 67 amino acids from the carboxy terminus, or any combinationof the above amino terminal and carboxy terminal deletions, of thecomplete amino acid sequence encoded by the cDNA clone contained inATCC™ Deposit No. 209198. Polypeptides encoded by these polynucleotidesare also encompassed by the invention. The present invention is alsodirected to nucleic acid molecules comprising, or alternatively,consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%,95%, 96%, 97%, 98%, 99% or 100% identical to the polynucleotide sequenceencoding the IL17RLP polypeptides described above. The present inventionalso encompasses the above polynucleotide sequences fused to aheterologous polynucleotide sequence. Polypeptides encoded by thesenucleic acids and/or polynucleotide sequences are also encompassed bythe invention, as are polypeptides comprising, or alternativelyconsisting of, an amino acid sequence at least 80%, 85%, 90%, 92%, 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequencedescribed above, and polynucleotides that encode such polypeptides.

As mentioned above, even if deletion of one or more amino acids from theN-terminus of a protein results in modification of loss of one or morebiological functions of the protein, other biological activities maystill be retained. Thus, the ability of the shortened IL17RLP mutein toinduce and/or bind to antibodies which recognize the complete or matureof the protein generally will be retained when less than the majority ofthe residues of the complete or mature protein are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that a IL17RLP mutein with a large number ofdeleted N-terminal amino acid residues may retain some biological orimmungenic activities. In fact, peptides composed of as few as sixIL17RLP amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the IL17RLPamino acid sequence shown in SEQ ID NO:2, up to the aspartic acidresidue at position number 421 and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising, or alternatively consisting of, the amino acid sequence ofresidues n²-426 of FIGS. 1A, 1B, and 1C (SEQ ID NO:2), where n² is aninteger in the range of 2 to 421, and 422 is the position of the firstresidue from the N-terminus of the complete IL17RLP polypeptide believedto be required for at least immunogenic activity of the IL17RLP protein.More in particular, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, a memberselected from the group consisting of the amino acid sequence ofresidues of S-2 to L-426; L-3 to L-426; V-4 to L-426; L-5 to L-426; L-6to L-426; S-7 to L-426; L-8 to L-426; A-9 to L-426; A-10 to L-426; L-11to L-426; C-12 to L-426; R-13 to L-426; S-14 to L-426; A-15 to L-426;V-16 to L-426; P-17 to L-426; R-18 to L-426; E-19 to L-426; P-20 toL-426; T-21 to L-426; V-22 to L-426; Q-23 to L-426; C-24 to L-426; G-25to L-426; S-26 to L-426; E-27 to L-426; T-28 to L-426; G-29 to L-426;P-30 to L-426; S-31 to L-426; P-32 to L-426; E-33 to L-426; W-34 toL-426; M-35 to L-426; L-36 to L-426; Q-37 to L-426; H-38 to L-426; D-39to L-426; L-40 to L-426; I-41 to L-426; P-42 to L-426; G-43 to L-426;D-44 to L-426; L-45 to L-426; R-46 to L-426; D-47 to L-426; L-48 toL-426; R-49 to L-426; V-50 to L-426; E-51 to L-426; P-52 to L-426; V-53to L-426; T-54 to L-426; T-55 to L-426; S-56 to L-426; V-57 to L-426;A-58 to L-426; T-59 to L-426; G-60 to L-426; D-61 to L-426; Y-62 toL-426; S-63 to L-426; I-64 to L-426; L-65 to L-426; M-66 to L-426; N-67to L-426; V-68 to L-426; S-69 to L-426; W-70 to L-426; V-71 to L-426;L-72 to L-426; R-73 to L-426; A-74 to L-426; D-75 to L-426; A-76 toL-426; S-77 to L-426; I-78 to L-426; R-79 to L-426; L-80 to L-426; L-81to L-426; K-82 to L-426; A-83 to L-426; T-84 to L-426; K-85 to L-426;I-86 to L-426; C-87 to L-426; V-88 to L-426; T-89 to L-426; G-90 toL-426; K-91 to L-426; S-92 to L-426; N-93 to L-426; F-94 to L-426; Q-95to L-426; S-96 to L-426; Y-97 to L-426; S-98 to L-426; C-99 to L-426;V-100 to L-426; R-101 to L-426; C-102 to L-426; N-103 to L-426; Y-104 toL-426; T-105 to L-426; E-106 to L-426; A-107 to L-426; F-108 to L-426;Q-109 to L-426; T-110 to L-426; Q-111 to L-426; T-112 to L-426; R-113 toL-426; P-114 to L-426; S-115 to L-426; G-116 to L-426; G-117 to L-426;K-118 to L-426; W-119 to L-426; T-120 to L-426; F-121 to L-426; S-122 toL-426; Y-123 to L-426; I-124 to L-426; G-125 to L-426; F-126 to L-426;P-127 to L-426; V-128 to L-426; E-129 to L-426; L-130 to L-426; N-131 toL-426; T-132 to L-426; V-133 to L-426; Y-134 to L-426; F-135 to L-426;I-136 to L-426; G-137 to L-426; A-138 to L-426; H-139 to L-426; N-140 toL-426; I-141 to L-426; P-142 to L-426; N-143 to L-426; A-144 to L-426;N-145 to L-426; M-146 to L-426; N-147 to L-426; E-148 to L-426; D-149 toL-426; G-150 to L-426; P-151 to L-426; S-152 to L-426; M-153 to L-426;S-154 to L-426; V-155 to L-426; N-156 to L-426; F-157 to L-426; T-158 toL-426; S-159 to L-426; P-160 to L-426; G-161 to L-426; C-162 to L-426;L-163 to L-426; D-164 to L-426; H-165 to L-426; I-166 to L-426; M-167 toL-426; K-168 to L-426; Y-169 to L-426; K-170 to L-426; K-171 to L-426;K-172 to L-426; C-173 to L-426; V-174 to L-426; K-175 to L-426; A-176 toL-426; G-177 to L-426; S-178 to L-426; L-179 to L-426; W-180 to L-426;D-181 to L-426; P-182 to L-426; N-183 to L-426; I-184 to L-426; T-185 toL-426; A-186 to L-426; C-187 to L-426; K-188 to L-426; K-189 to L-426;N-190 to L-426; E-191 to L-426; E-192 to L-426; T-193 to L-426; V-194 toL-426; E-195 to L-426; V-196 to L-426; N-197 to L-426; F-198 to L-426;T-199 to L-426; T-200 to L-426; T-201 to L-426; P-202 to L-426; L-203 toL-426; G-204 to L-426; N-205 to L-426; R-206 to L-426; Y-207 to L-426;M-208 to L-426; A-209 to L-426; L-210 to L-426; I-211 to L-426; Q-212 toL-426; H-213 to L-426; S-214 to L-426; T-215 to L-426; I-216 to L-426;I-217 to L-426; G-218 to L-426; F-219 to L-426; S-220 to L-426; Q-221 toL-426; V-222 to L-426; F-223 to L-426; E-224 to L-426; P-225 to L-426;H-226 to L-426; Q-227 to L-426; K-228 to L-426; K-229 to L-426; Q-230 toL-426; T-231 to L-426; R-232 to L-426; A-233 to L-426; S-234 to L-426;V-235 to L-426; V-236 to L-426; I-237 to L-426; P-238 to L-426; V-239 toL-426; T-240 to L-426; G-241 to L-426; D-242 to L-426; S-243 to L-426;E-244 to L-426; G-245 to L-426; A-246 to L-426; T-247 to L-426; V-248 toL-426; Q-249 to L-426; L-250 to L-426; T-251 to L-426; P-252 to L-426;Y-253 to L-426; F-254 to L-426; P-255 to L-426; T-256 to L-426; C-257 toL-426; G-258 to L-426; S-259 to L-426; D-260 to L-426; C-261 to L-426;I-262 to L-426; R-263 to L-426; H-264 to L-426; K-265 to L-426; G-266 toL-426; T-267 to L-426; V-268 to L-426; V-269 to L-426; L-270 to L-426;C-271 to L-426; P-272 to L-426; Q-273 to L-426; T-274 to L-426; G-275 toL-426; V-276 to L-426; P-277 to L-426; F-278 to L-426; P-279 to L-426;L-280 to L-426; D-281 to L-426; N-282 to L-426; N-283 to L-426; K-284 toL-426; S-285 to L-426; K-286 to L-426; P-287 to L-426; G-288 to L-426;G-289 to L-426; W-290 to L-426; L-291 to L-426; P-292 to L-426; L-293 toL-426; L-294 to L-426; L-295 to L-426; L-296 to L-426; S-297 to L-426;L-298 to L-426; L-299 to L-426; V-300 to L-426; A-301 to L-426; T-302 toL-426; W-303 to L-426; V-304 to L-426; L-305 to L-426; V-306 to L-426;A-307 to L-426; G-308 to L-426; I-309 to L-426; Y-310 to L-426; L-311 toL-426; M-312 to L-426; W-313 to L-426; R-314 to L-426; H-315 to L-426;E-316 to L-426; R-317 to L-426; I-318 to L-426; K-319 to L-426; K-320 toL-426; T-321 to L-426; S-322 to L-426; F-323 to L-426; S-324 to L-426;T-325 to L-426; T-326 to L-426; T-327 to L-426; L-328 to L-426; L-329 toL-426; P-330 to L-426; P-331 to L-426; I-332 to L-426; K-333 to L-426;V-334 to L-426; L-335 to L-426; V-336 to L-426; V-337 to L-426; Y-338 toL-426; P-339 to L-426; S-340 to L-426; E-341 to L-426; I-342 to L-426;C-343 to L-426; F-344 to L-426; H-345 to L-426; H-346 to L-426; T-347 toL-426; I-348 to L-426; C-349 to L-426; Y-350 to L-426; F-351 to L-426;T-352 to L-426; E-353 to L-426; F-354 to L-426; L-355 to L-426; Q-356 toL-426; N-357 to L-426; H-358 to L-426; C-359 to L-426; R-360 to L-426;S-361 to L-426; E-362 to L-426; V-363 to L-426; I-364 to L-426; L-365 toL-426; E-366 to L-426; K-367 to L-426; W-368 to L-426; Q-369 to L-426;K-370 to L-426; K-371 to L-426; K-372 to L-426; I-373 to L-426; A-374 toL-426; E-375 to L-426; M-376 to L-426; G-377 to L-426; P-378 to L-426;V-379 to L-426; Q-380 to L-426; W-381 to L-426; L-382 to L-426; A-383 toL-426; T-384 to L-426; Q-385 to L-426; K-386 to L-426; K-387 to L-426;A-388 to L-426; A-389 to L-426; D-390 to L-426; K-391 to L-426; V-392 toL-426; V-393 to L-426; F-394 to L-426; L-395 to L-426; L-396 to L-426;S-397 to L-426; N-398 to L-426; D-399 to L-426; V-400 to L-426; N-401 toL-426; S-402 to L-426; V-403 to L-426; C-404 to L-426; D-405 to L-426;G-406 to L-426; T-407 to L-426; C-408 to L-426; G-409 to L-426; K-410 toL-426; S-411 to L-426; E-412 to L-426; G-413 to L-426; S-414 to L-426;P-415 to L-426; S-416 to L-426; E-417 to L-426; N-418 to L-426; S-419 toL-426; Q-420 to L-426; and D-421 to L-426 of the IL17RLP amino acidsequence shown in FIGS. 1A, 1B, and 1C (which is identical to thesequence shown as SEQ ID NO:2, with the exception that the amino acidresidues in FIGS. 1A, 1B, and 1C are numbered consecutively from 1through 426 from the N-terminus to the C-terminus, while the amino acidresidues in SEQ ID NO:2 are numbered consecutively from −19 through 407to reflect the position of the predicted signal peptide). Polypeptidesencoded by these polynucleotides are also encompassed by the invention.The present invention is also directed to nucleic acid moleculescomprising, or alternatively, consisting of, a polynucleotide sequenceat least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identicalto the polynucleotide sequence encoding the IL17RLP polypeptidesdescribed above. The present invention also encompasses the abovepolynucleotide sequences fused to a heterologous polynucleotidesequence. Polypeptides encoded by these nucleic acids and/orpolynucleotide sequences are also encompassed by the invention, as arepolypeptides comprising, or alternatively consisting of, an amino acidsequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence described above, andpolynucleotides that encode such polypeptides.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other biological activitiesmay still be retained. Thus, the ability of the shortened IL17RLP muteinto induce and/or bind to antibodies which recognize the complete ormature of the protein generally will be retained when less than themajority of the residues of the complete or mature protein are removedfrom the C-terminus. Whether a particular polypeptide lacking C-terminalresidues of a complete protein retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a IL17RLP mutein with a largenumber of deleted C-terminal amino acid residues may retain somebiological or immungenic activities. In fact, peptides composed of asfew as six IL17RLP amino acid residues may often evoke an immuneresponse.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the IL17RLP shown in SEQ ID NO:2, up to the leucine residueat position number 6, and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising, oralternatively consisting of, the amino acid sequence of residues 1-m² ofSEQ ID NO:2, where m² is an integer in the range of 6 to 426, and 6 isthe position of the first residue from the C-terminus of the completeIL17RLP polypeptide believed to be required for at least immunogenicactivity of the IL17RLP protein. More in particular, the inventionprovides polynucleotides encoding polypeptides comprising, oralternatively consisting of, a member selected from the group consistingof the amino acid sequence of residues M-1 to C-425; M-1 to P-424; M-1to S-423; M-1 to S-422; M-1 to D-421; M-1 to Q-420; M-1 to S-419; M-1 toN-418; M-1 to E-417; M-1 to S-416; M-1 to P-415; M-1 to S-414; M-1 toG-413; M-1 to E-412; M-1 to S-411; M-1 to K-410; M-1 to G-409; M-1 toC-408; M-1 to T-407; M-1 to G-406; M-1 to D-405; M-1 to C-404; M-1 toV-403; M-1 to S-402; M-1 to N-401; M-1 to V-400; M-1 to D-399; M-1 toN-398; M-1 to S-397; M-1 to L-396; M-1 to L-395; M-1 to F-394; M-1 toV-393; M-1 to V-392; M-1 to K-391; M-1 to D-390; M-1 to A-389; M-1 toA-388; M-1 to K-387; M-1 to K-386; M-1 to Q-385; M-1 to T-384; M-1 toA-383; M-1 to L-382; M-1 to W-381; M-1 to Q-380; M-1 to V-379; M-1 toP-378; M-1 to G-377; M-1 to M-376; M-1 to E-375; M-1 to A-374; M-1 toI-373; M-1 to K-372; M-1 to K-371; M-1 to K-370; M-1 to Q-369; M-1 toW-368; M-1 to K-367; M-1 to E-366; M-1 to L-365; M-1 to I-364; M-1 toV-363; M-1 to E-362; M-1 to S-361; M-1 to R-360; M-1 to C-359; M-1 toH-358; M-1 to N-357; M-1 to Q-356; M-1 to L-355; M-1 to F-354; M-1 toE-353; M-1 to T-352; M-1 to F-351; M-1 to Y-350; M-1 to C-349; M-1 toI-348; M-1 to T-347; M-1 to H-346; M-1 to H-345; M-1 to F-344; M-1 toC-343; M-1 to I-342; M-1 to E-341; M-1 to S-340; M-1 to P-339; M-1 toY-338; M-1 to V-337; M-1 to V-336; M-1 to L-335; M-1 to V-334; M-1 toK-333; M-1 to I-332; M-1 to P-331; M-1 to P-330; M-1 to L-329; M-1 toL-328; M-1 to T-327; M-1 to T-326; M-1 to T-325; M-1 to S-324; M-1 toF-323; M-1 to S-322; M-1 to T-321; M-1 to K-320; M-1 to K-319; M-1 toI-318; M-1 to R-317; M-1 to E-316; M-1 to H-315; M-1 to R-314; M-1 toW-313; M-1 to M-312; M-1 to L-311; M-1 to Y-310; M-1 to I-309; M-1 toG-308; M-1 to A-307; M-1 to V-306; M-1 to L-305; M-1 to V-304; M-1 toW-303; M-1 to T-302; M-1 to A-301; M-1 to V-300; M-1 to L-299; M-1 toL-298; M-1 to S-297; M-1 to L-296; M-1 to L-295; M-1 to L-294; M-1 toL-293; M-1 to P-292; M-1 to L-291; M-1 to W-290; M-1 to G-289; M-1 toG-288; M-1 to P-287; M-1 to K-286; M-1 to S-285; M-1 to K-284; M-1 toN-283; M-1 to N-282; M-1 to D-281; M-1 to L-280; M-1 to P-279; M-1 toF-278; M-1 to P-277; M-1 to V-276; M-1 to G-275; M-1 to T-274; M-1 toQ-273; M-1 to P-272; M-1 to C-271; M-1 to L-270; M-1 to V-269; M-1 toV-268; M-1 to T-267; M-1 to G-266; M-1 to K-265; M-1 to H-264; M-1 toR-263; M-1 to I-262; M-1 to C-261; M-1 to D-260; M-1 to S-259; M-1 toG-258; M-1 to C-257; M-1 to T-256; M-1 to P-255; M-1 to F-254; M-1 toY-253; M-1 to P-252; M-1 to T-251; M-1 to L-250; M-1 to Q-249; M-1 toV-248; M-1 to T-247; M-1 to A-246; M-1 to G-245; M-1 to E-244; M-1 toS-243; M-1 to D-242; M-1 to G-241; M-1 to T-240; M-1 to V-239; M-1 toP-238; M-1 to I-237; M-1 to V-236; M-1 to V-235; M-1 to S-234; M-1 toA-233; M-1 to R-232; M-1 to T-231; M-1 to Q-230; M-1 to K-229; M-1 toK-228; M-1 to Q-227; M-1 to H-226; M-1 to P-225; M-1 to E-224; M-1 toF-223; M-1 to V-222; M-1 to Q-221; M-1 to S-220; M-1 to F-219; M-1 toG-218; M-1 to I-217; M-1 to I-216; M-1 to T-215; M-1 to S-214; M-1 toH-213; M-1 to Q-212; M-1 to I-211; M-1 to L-210; M-1 to A-209; M-1 toM-208; M-1 to Y-207; M-1 to R-206; M-1 to N-205; M-1 to G-204; M-1 toL-203; M-1 to P-202; M-1 to T-201; M-1 to T-200; M-1 to T-199; M-1 toF-198; M-1 to N-197; M-1 to V-196; M-1 to E-195; M-1 to V-194; M-1 toT-193; M-1 to E-192; M-1 to E-191; M-1 to N-190; M-1 to K-189; M-1 toK-188; M-1 to C-187; M-1 to A-186; M-1 to T-185; M-1 to T-184; M-1 toN-183; M-1 to P-182; M-1 to D-181; M-1 to W-180; M-1 to L-179; M-1 toS-178; M-1 to G-177; M-1 to A-176; M-1 to K-175; M-1 to V-174; M-1 toC-173; M-1 to K-172; M-1 to K-171; M-1 to K-170; M-1 to Y-169; M-1 toK-168; M-1 to M-167; M-1 to I-166; M-1 to H-165; M-1 to D-164; M-1 toL-163; M-1 to C-162; M-1 to G-161; M-1 to P-160; M-1 to S-159; M-1 toT-158; M-1 to F-157; M-1 to N-156; M-1 to V-155; M-1 to S-154; M-1 toM-153; M-1 to S-152; M-1 to P-151; M-1 to G-150; M-1 to D-149; M-1 toE-148; M-1 to N-147; M-1 to M-146; M-1 to N-145; M-1 to A-144; M-1 toN-143; M-1 to P-142; M-1 to I-141; M-1 to N-140; M-1 to H-139; M-1 toA-138; M-1 to G-137; M-1 to I-136; M-1 to F-135; M-1 to Y-134; M-1 toV-133; M-1 to T-132; M-1 to N-131; M-1 to L-130; M-1 to E-129; M-1 toV-128; M-1 to P-127; M-1 to F-126; M-1 to G-125; M-1 to I-124; M-1 toY-123; M-1 to S-122; M-1 to F-121; M-1 to T-120; M-1 to W-119; M-1 toK-118; M-1 to G-117; M-1 to G-116; M-1 to S-115; M-1 to P-114; M-1 toR-113; M-1 to T-112; M-1 to Q-111; M-1 to T-110; M-1 to Q-109; M-1 toF-108; M-1 to A-107; M-1 to E-106; M-1 to T-105; M-1 to Y-104; M-1 toN-103; M-1 to C-102; M-1 to R-101; M-1 to V-100; M-1 to C-99; M-1 toS-98; M-1 to Y-97; M-1 to S-96; M-1 to Q-95; M-1 to F-94; M-1 to N-93;M-1 to S-92; M-1 to K-91; M-1 to G-90; M-1 to T-89; M-1 to V-88; M-1 toC-87; M-1 to I-86; M-1 to K-85; M-1 to T-84; M-1 to A-83; M-1 to K-82;M-1 to L-81; M-1 to L-80; M-1 to R-79; M-1 to I-78; M-1 to S-77; M-1 toA-76; M-1 to D-75; M-1 to A-74; M-1 to R-73; M-1 to L-72; M-1 to V-71;M-1 to W-70; M-1 to S-69; M-1 to V-68; M-1 to N-67; M-1 to M-66; M-1 toL-65; M-1 to I-64; M-1 to S-63; M-1 to Y-62; M-1 to D-61; M-1 to G-60;M-1 to T-59; M-1 to A-58; M-1 to V-57; M-1 to S-56; M-1 to T-55; M-1 toT-54; M-1 to V-53; M-1 to P-52; M-1 to E-51; M-1 to V-50; M-1 to R-49;M-1 to L-48; M-1 to D-47; M-1 to R-46; M-1 to L-45; M-1 to D-44; M-1 toG-43; M-1 to P-42; M-1 to I-41; M-1 to L-40; M-1 to D-39; M-1 to H-38;M-1 to Q-37; M-1 to L-36; M-1 to M-35; M-1 to W-34; M-1 to E-33; M-1 toP-32; M-1 to S-31; M-1 to P-30; M-1 to G-29; M-1 to T-28; M-1 to E-27;M-1 to S-26; M-1 to G-25; M-1 to C-24; M-1 to Q-23; M-1 to V-22; M-1 toT-21; M-1 to P-20; M-1 to E-19; M-1 to R-18; M-1 to P-17; M-1 to V-16;M-1 to A-15; M-1 to S-14; M-1 to R-13; M-1 to C-12; M-1 to L-11; M-1 toA-10; M-1 to A-9; M-1 to L-8; M-1 to S-7; and M-1 to L-6 of the sequenceof the IL17RLP sequence shown in FIGS. 1A, 1B, and 1C (which isidentical to the sequence shown as SEQ ID NO:2, with the exception thatthe amino acid residues in FIGS. 1A, 1B, and 1C are numberedconsecutively from 1 through 426 from the N-terminus to the C-terminus,while the amino acid residues in SEQ ID NO:2 are numbered consecutivelyfrom −19 through 407 to reflect the position of the predicted signalpeptide). Polypeptides encoded by these polynucleotides are alsoencompassed by the invention. The present invention is also directed tonucleic acid molecules comprising, or alternatively, consisting of, apolynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%,99% or 100% identical to the polynucleotide sequence encoding theIL17RLP polypeptides described above. The present invention alsoencompasses the above polynucleotide sequences fused to a heterologouspolynucleotide sequence. Polypeptides encoded by these nucleic acidsand/or polynucleotide sequences are also encompassed by the invention,as are polypeptides comprising, or alternatively consisting of, an aminoacid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence described above, andpolynucleotides that encode such polypeptides.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini of an IL17RLPpolypeptide, which may be described generally as comprising, oralternatively consisting of, residues n²-m² of FIGS. 1A, 1B, and 1C (SEQID NO:2), where n² and m² are integers as described above.

Also as mentioned above, even if deletion of one or more amino acidsfrom the N-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other biological activitiesmay still be retained. Thus, the ability of the shortened extracellulardomain of the IL17RLP mutein to induce and/or bind to antibodies whichrecognize the extracellular domain of the IL17RLP protein generally willbe retained when less than the majority of the residues of theextracellular domain of the IL17RLP protein are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof an extracellular domain of the IL17RLP protein retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thata IL17RLP mutein with a large number of deleted N-terminal amino acidresidues may retain some biological or immunogenic activities. In fact,peptides composed of as few as six amino acid residues of theextracellular domain of the IL17RLP protein may often evoke an immuneresponse.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of theextracellular domain of the IL17RLP amino acid sequence shown in SEQ IDNO:2, up to the aspartic acid residue at position number 421 andpolynucleotides encoding such polypeptides. In particular, the presentinvention provides polypeptides comprising, or alternatively consistingof, the amino acid sequence of residues n³-290 of FIGS. 1A, 1B, and 1C(SEQ ID NO:2), where n³ is an integer in the range of 15 to 285, and 286is the position of the first residue from the N-terminus of theextracellular domain of the IL17RLP polypeptide believed to be requiredfor at least immunogenic activity of the IL17RLP protein. More inparticular, the invention provides polynucleotides encoding polypeptidescomprising, or alternatively consisting of, a member selected from thegroup consisting of the amino acid sequence of residues of A-15 toW-290; V-16 to W-290; P-17 to W-290; R-18 to W-290; E-19 to W-290; P-20to W-290; T-21 to W-290; V-22 to W-290; Q-23 to W-290; C-24 to W-290;G-25 to W-290; S-26 to W-290; E-27 to W-290; T-28 to W-290; G-29 toW-290; P-30 to W-290; S-31 to W-290; P-32 to W-290; E-33 to W-290; W-34to W-290; M-35 to W-290; L-36 to W-290; Q-37 to W-290; H-38 to W-290;D-39 to W-290; L-40 to W-290; I-41 to W-290; P-42 to W-290; G-43 toW-290; D-44 to W-290; L-45 to W-290; R-46 to W-290; D-47 to W-290; L-48to W-290; R-49 to W-290; V-50 to W-290; E-51 to W-290; P-52 to W-290;V-53 to W-290; T-54 to W-290; T-55 to W-290; S-56 to W-290; V-57 toW-290; A-58 to W-290; T-59 to W-290; G-60 to W-290; D-61 to W-290; Y-62to W-290; S-63 to W-290; I-64 to W-290; L-65 to W-290; M-66 to W-290;N-67 to W-290; V-68 to W-290; S-69 to W-290; W-70 to W-290; V-71 toW-290; L-72 to W-290; R-73 to W-290; A-74 to W-290; D-75 to W-290; A-76to W-290; S-77 to W-290; I-78 to W-290; R-79 to W-290; L-80 to W-290;L-81 to W-290; K-82 to W-290; A-83 to W-290; T-84 to W-290; K-85 toW-290; I-86 to W-290; C-87 to W-290; V-88 to W-290; T-89 to W-290; G-90to W-290; K-91 to W-290; S-92 to W-290; N-93 to W-290; F-94 to W-290;Q-95 to W-290; S-96 to W-290; Y-97 to W-290; S-98 to W-290; C-99 toW-290; V-100 to W-290; R-101 to W-290; C-102 to W-290; N-103 to W-290;Y-104 to W-290; T-105 to W-290; E-106 to W-290; A-107 to W-290; F-108 toW-290; Q-109 to W-290; T-110 to W-290; Q-111 to W-290; T-112 to W-290;R-113 to W-290; P-114 to W-290; S-115 to W-290; G-116 to W-290; G-117 toW-290; K-118 to W-290; W-119 to W-290; T-120 to W-290; F-121 to W-290;S-122 to W-290; Y-123 to W-290; I-124 to W-290; G-125 to W-290; F-126 toW-290; P-127 to W-290; V-128 to W-290; E-129 to W-290; L-130 to W-290;N-131 to W-290; T-132 to W-290; V-133 to W-290; Y-134 to W-290; F-135 toW-290; I-136 to W-290; G-137 to W-290; A-138 to W-290; H-139 to W-290;N-140 to W-290; I-141 to W-290; P-142 to W-290; N-143 to W-290; A-144 toW-290; N-145 to W-290; M-146 to W-290; N-147 to W-290; E-148 to W-290;D-149 to W-290; G-150 to W-290; P-151 to W-290; S-152 to W-290; M-153 toW-290; S-154 to W-290; V-155 to W-290; N-156 to W-290; F-157 to W-290;T-158 to W-290; S-159 to W-290; P-160 to W-290; G-161 to W-290; C-162 toW-290; L-163 to W-290; D-164 to W-290; H-165 to W-290; I-166 to W-290;M-167 to W-290; K-168 to W-290; Y-169 to W-290; K-170 to W-290; K-171 toW-290; K-172 to W-290; C-173 to W-290; V-174 to W-290; K-175 to W-290;A-176 to W-290; G-177 to W-290; S-178 to W-290; L-179 to W-290; W-180 toW-290; D-181 to W-290; P-182 to W-290; N-183 to W-290; I-184 to W-290;T-185 to W-290; A-186 to W-290; C-187 to W-290; K-188 to W-290; K-189 toW-290; N-190 to W-290; E-191 to W-290; E-192 to W-290; T-193 to W-290;V-194 to W-290; E-195 to W-290; V-196 to W-290; N-197 to W-290; F-198 toW-290; T-199 to W-290; T-200 to W-290; T-201 to W-290; P-202 to W-290;L-203 to W-290; G-204 to W-290; N-205 to W-290; R-206 to W-290; Y-207 toW-290; M-208 to W-290; A-209 to W-290; L-210 to W-290; I-211 to W-290;Q-212 to W-290; H-213 to W-290; S-214 to W-290; T-215 to W-290; I-216 toW-290; I-217 to W-290; G-218 to W-290; F-219 to W-290; S-220 to W-290;Q-221 to W-290; V-222 to W-290; F-223 to W-290; E-224 to W-290; P-225 toW-290; H-226 to W-290; Q-227 to W-290; K-228 to W-290; K-229 to W-290;Q-230 to W-290; T-231 to W-290; R-232 to W-290; A-233 to W-290; S-234 toW-290; V-235 to W-290; V-236 to W-290; I-237 to W-290; P-238 to W-290;V-239 to W-290; T-240 to W-290; G-241 to W-290; D-242 to W-290; S-243 toW-290; E-244 to W-290; G-245 to W-290; A-246 to W-290; T-247 to W-290;V-248 to W-290; Q-249 to W-290; L-250 to W-290; T-251 to W-290; P-252 toW-290; Y-253 to W-290; F-254 to W-290; P-255 to W-290; T-256 to W-290;C-257 to W-290; G-258 to W-290; S-259 to W-290; D-260 to W-290; C-261 toW-290; I-262 to W-290; R-263 to W-290; H-264 to W-290; K-265 to W-290;G-266 to W-290; T-267 to W-290; V-268 to W-290; V-269 to W-290; L-270 toW-290; C-271 to W-290; P-272 to W-290; Q-273 to W-290; T-274 to W-290;G-275 to W-290; V-276 to W-290; P-277 to W-290; F-278 to W-290; P-279 toW-290; L-280 to W-290; D-281 to W-290; N-282 to W-290; N-283 to W-290;K-284 to W-290; and S-285 to W-290 of the IL17RLP amino acid sequenceshown in FIGS. 1A, 1B, and 1C (which is identical to the sequence shownas SEQ ID NO:2, with the exception that the amino acid residues in FIGS.1A, 1B, and 1C are numbered consecutively from 1 through 426 from theN-terminus to the C-terminus, while the amino acid residues in SEQ IDNO:2 are numbered consecutively from −19 through 407 to reflect theposition of the predicted signal peptide). Polypeptides encoded by thesepolynucleotides are also encompassed by the invention. The presentinvention is also directed to nucleic acid molecules comprising, oralternatively, consisting of, a polynucleotide sequence at least 80%,85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to thepolynucleotide sequence encoding the IL17RLP polypeptides describedabove. The present invention also encompasses the above polynucleotidesequences fused to a heterologous polynucleotide sequence. Polypeptidesencoded by these nucleic acids and/or polynucleotide sequences are alsoencompassed by the invention, as are polypeptides comprising, oralternatively consisting of, an amino acid sequence at least 80%, 85%,90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence described above, and polynucleotides that encode suchpolypeptides.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of the extracellular domain of an IL17RLP proteinresults in modification of loss of one or more biological functions ofthe protein, other biological activities may still be retained. Thus,the ability of the shortened extracellular domain of an IL17RLP muteinto induce and/or bind to antibodies which recognize the extracellulardomain of an IL17RLP protein generally will be retained when less thanthe majority of the residues of the extracellular domain of an IL17RLPprotein are removed from the C-terminus. Whether a particularpolypeptide lacking C-terminal residues of a extracellular domain of anIL17RLP protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that an extracellular domain of an IL17RLPmutein with a large number of deleted C-terminal amino acid residues mayretain some biological or immungenic activities. In fact, peptidescomposed of as few as six extracellular IL17RLP amino acid residues mayoften evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the extracellular domain of the IL17RLP shown in SEQ IDNO:2, up to the leucine residue at position number 6, andpolynucleotides encoding such polypeptides. In particular, the presentinvention provides polypeptides comprising, or alternatively consistingof, the amino acid sequence of residues 15-m³ of SEQ ID NO:2, where m³is an integer in the range of 20 to 290, and 20 is the position of thefirst residue from the C-terminus of the extracellular domain IL17RLPpolypeptide believed to be required for at least immunogenic activity ofthe IL17RLP protein. More in particular, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, a member selected from the group consisting of the aminoacid sequence of residues A-15 to W-290; A-15 to G-289; A-15 to G-288;A-15 to P-287; A-15 to K-286; A-15 to S-285; A-15 to K-284; A-15 toN-283; A-15 to N-282; A-15 to D-281; A-15 to L-280; A-15 to P-279; A-15to F-278; A-15 to P-277; A-15 to V-276; A-15 to G-275; A-15 to T-274;A-15 to Q-273; A-15 to P-272; A-15 to C-271; A-15 to L-270; A-15 toV-269; A-15 to V-268; A-15 to T-267; A-15 to G-266; A-15 to K-265; A-15to H-264; A-15 to R-263; A-15 to I-262; A-15 to C-261; A-15 to D-260;A-15 to S-259; A-15 to G-258; A-15 to C-257; A-15 to T-256; A-15 toP-255; A-15 to F-254; A-15 to Y-253; A-15 to P-252; A-15 to T-251; A-15to L-250; A-15 to Q-249; A-15 to V-248; A-15 to T-247; A-15 to A-246;A-15 to G-245; A-15 to E-244; A-15 to S-243; A-15 to D-242; A-15 toG-241; A-15 to T-240; A-15 to V-239; A-15 to P-238; A-15 to I-237; A-15to V-236; A-15 to V-235; A-15 to S-234; A-15 to A-233; A-15 to R-232;A-15 to T-231; A-15 to Q-230; A-15 to K-229; A-15 to K-228; A-15 toQ-227; A-15 to H-226; A-15 to P-225; A-15 to E-224; A-15 to F-223; A-15to V-222; A-15 to Q-221; A-15 to S-220; A-15 to F-219; A-15 to G-218;A-15 to I-217; A-15 to I-216; A-15 to T-215; A-15 to S-214; A-15 toH-213; A-15 to Q-212; A-15 to I-211; A-15 to L-210; A-15 to A-209; A-15to M-208; A-15 to Y-207; A-15 to R-206; A-15 to N-205; A-15 to G-204;A-15 to L-203; A-15 to P-202; A-15 to T-201; A-15 to T-200; A-15 toT-199; A-15 to F-198; A-15 to N-197; A-15 to V-196; A-15 to E-195; A-15to V-194; A-15 to T-193; A-15 to E-192; A-15 to E-191; A-15 to N-190;A-15 to K-189; A-15 to K-188; A-15 to C-187; A-15 to A-186; A-15 toT-185; A-15 to I-184; A-15 to N-183; A-15 to P-182; A-15 to D-181; A-15to W-180; A-15 to L-179; A-15 to S-178; A-15 to G-177; A-15 to A-176;A-15 to K-175; A-15 to V-174; A-15 to C-173; A-15 to K-172; A-15 toK-171; A-15 to K-170; A-15 to Y-169; A-15 to K-168; A-15 to A-1567; A-15to I-166; A-15 to H-165; A-15 to D-164; A-15 to L-163; A-15 to C-162;A-15 to G-161; A-15 to P-160; A-15 to S-159; A-15 to T-158; A-15 toF-157; A-15 to N-156; A-15 to V-155; A-15 to S-154; A-15 to A-1553; A-15to S-152; A-15 to P-151; A-15 to G-150; A-15 to D-149; A-15 to E-148;A-15 to N-147; A-15 to A-1546; A-15 to N-145; A-15 to A-144; A-15 toN-143; A-15 to P-142; A-15 to I-141; A-15 to N-140; A-15 to H-139; A-15to A-138; A-15 to G-137; A-15 to I-136; A-15 to F-135; A-15 to Y-134;A-15 to V-133; A-15 to T-132; A-15 to N-131; A-15 to L-130; A-15 toE-129; A-15 to V-128; A-15 to P-127; A-15 to F-126; A-15 to G-125; A-15to I-124; A-15 to Y-123; A-15 to S-122; A-15 to F-121; A-15 to T-120;A-15 to W-119; A-15 to K-118; A-15 to G-117; A-15 to G-116; A-15 toS-115; A-15 to P-114; A-15 to R-113; A-15 to T-112; A-15 to Q-111; A-15to T-110; A-15 to Q-109; A-15 to F-108; A-15 to A-107; A-15 to E-106;A-15 to T-105; A-15 to Y-104; A-15 to N-103; A-15 to C-102; A-15 toR-101; A-15 to V-100; A-15 to C-99; A-15 to S-98; A-15 to Y-97; A-15 toS-96; A-15 to Q-95; A-15 to F-94; A-15 to N-93; A-15 to S-92; A-15 toK-91; A-15 to G-90; A-15 to T-89; A-15 to V-88; A-15 to C-87; A-15 toI-86; A-15 to K-85; A-15 to T-84; A-15 to A-83; A-15 to K-82; A-15 toL-81; A-15 to L-80; A-15 to R-79; A-15 to I-78; A-15 to S-77; A-15 toA-76; A-15 to D-75; A-15 to A-74; A-15 to R-73; A-15 to L-72; A-15 toV-71; A-15 to W-70; A-15 to S-69; A-15 to V-68; A-15 to N-67; A-15 toM-66; A-15 to L-65; A-15 to I-64; A-15 to S-63; A-15 to Y-62; A-15 toD-61; A-15 to G-60; A-15 to T-59; A-15 to A-58; A-15 to V-57; A-15 toS-56; A-15 to T-55; A-15 to T-54; A-15 to V-53; A-15 to P-52; A-15 toE-51; A-15 to V-50; A-15 to R-49; A-15 to L-48; A-15 to D-47; A-15 toR-46; A-15 to L-45; A-15 to D-44; A-15 to G-43; A-15 to P-42; A-15 toI-41; A-15 to L-40; A-15 to D-39; A-15 to H-38; A-15 to Q-37; A-15 toL-36; A-15 to M-35; A-15 to W-34; A-15 to E-33; A-15 to P-32; A-15 toS-31; A-15 to P-30; A-15 to G-29; A-15 to T-28; A-15 to E-27; A-15 toS-26; A-15 to G-25; A-15 to C-24; A-15 to Q-23; A-15 to V-22; A-15 toT-21; and A-15 to P-20 of the sequence of the IL17RLP sequence shown inFIGS. 1A, 1B, and 1C (which is identical to the sequence shown as SEQ IDNO:2, with the exception that the amino acid residues in FIGS. 1A, 1B,and 1C are numbered consecutively from 1 through 426 from the N-terminusto the C-terminus, while the amino acid residues in SEQ ID NO:2 arenumbered consecutively from −19 through 407 to reflect the position ofthe predicted signal peptide). Polypeptides encoded by thesepolynucleotides are also encompassed by the invention. The presentinvention is also directed to nucleic acid molecules comprising, oralternatively, consisting of, a polynucleotide sequence at least 80%,85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to thepolynucleotide sequence encoding the IL17RLP polypeptides describedabove. The present invention also encompasses the above polynucleotidesequences fused to a heterologous polynucleotide sequence. Polypeptidesencoded by these nucleic acids and/or polynucleotide sequences are alsoencompassed by the invention, as are polypeptides comprising, oralternatively consisting of, an amino acid sequence at least 80%, 85%,90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence described above, and polynucleotides that encode suchpolypeptides.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini of an extracellulardomain of the IL17RLP polypeptide, which may be described generally ascomprising, or alternatively consisting of, residues n³-m³ of FIGS. 1A,1B, and 1C (SEQ ID NO:2), where n³ and m³ are integers as describedabove.

One specific embodiment of the present invention includes polypeptidefragments of the amino acid sequence set forth in SEQ ID NO:2 which maybe used, for example, to generate monoclonal antibodies as describedherein below. Particular examples of such polypeptides includepolypeptides comprising, or alternatively consisting of, the amino acidsequences PREPTVQCGSETGPSPE (SEQ ID NO:14) (i.e., amino acid positionsPro-17 to Glu-33 of SEQ ID NO:2); LDHIMKYKKK (SEQ ID NO:15) (i.e., aminoacid positions Leu-163 to Lys-173 of SEQ ID NO:2); and KKNEETVEVN (SEQID NO:16) (i.e., amino acid positions Lys-188 to Asn-197 of SEQ IDNO:2).

In addition to terminal deletion forms of the protein discussed above,it also will be recognized by one of ordinary skill in the art that someamino acid sequences of the IL17RLP polypeptide can be varied withoutsignificant effect of the structure or function of the protein. If suchdifferences in sequence are contemplated, it should be remembered thatthere will be critical areas on the protein which determine activity.

Thus, the invention further includes variations of the IL17RLPpolypeptide which show substantial IL17RLP polypeptide activity or whichinclude regions of IL17RLP protein such as the protein portionsdiscussed below. Such mutants include deletions, insertions, inversions,repeats, and type substitutions selected according to general rulesknown in the art so as have little effect on activity. For example,guidance concerning how to make phenotypically silent amino acidsubstitutions is provided wherein the authors indicate that there aretwo main approaches for studying the tolerance of an amino acid sequenceto change (Bowie, J. U., et al., Science 247:1306-1310 (1990)). Thefirst method relies on the process of evolution, in which mutations areeither accepted or rejected by natural selection. The second approachuses genetic engineering to introduce amino acid changes at specificpositions of a cloned gene and selections or screens to identifysequences that maintain functionality.

As the authors state, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which amino acid changes are likely to be permissive at acertain position of the protein. For example, most buried amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Other such phenotypically silentsubstitutions are described by Bowie and coworkers (supra) and thereferences cited therein. Typically seen as conservative substitutionsare the replacements, one for another, among the aliphatic amino acidsAla, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Thus, the fragment, derivative or analog of the polypeptide of SEQ IDNO:2, or that encoded by the deposited cDNA, may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the extracellular domain ofthe polypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or (v) one in which the additional amino acids are fused to theabove form of the polypeptide, such as an IgG Fc fusion region peptideor leader or secretory sequence or a sequence which is employed forpurification of the above form of the polypeptide or a proproteinsequence. Such fragments, derivatives and analogs are deemed to bewithin the scope of those skilled in the art from the teachings herein.

Thus, the IL17RLP of the present invention may include one or more aminoacid substitutions, deletions or additions, either from naturalmutations or human manipulation. As indicated, changes are preferably ofa minor nature, such as conservative amino acid substitutions that donot significantly affect the folding or activity of the protein (seeTable II).

TABLE II Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

For example, site directed changes at the amino acid level of IL17RLPcan be made by replacing a particular amino acid with a conservativeamino acid. Preferred conservative mutations include: M1 replaced withA, G, I, L, S, T, or V; S2 replaced with A, G, I, L, T, M, or V; L3replaced with A, G, I, S, T, M, or V; V4 replaced with A, G, I, L, S, T,or M; L5 replaced with A, G, I, S, T, M, or V; L6 replaced with A, G, I,S, T, M, or V; S7 replaced with A, G, I, L, T, M, or V; L8 replaced withA, G, I, S, T, M, or V; A9 replaced with G, I, L, S, T, M, or V; A10replaced with G, I, L, S, T, M, or V; L11 replaced with A, G, I, S, T,M, or V; R13 replaced with H, or K; S14 replaced with A, G, I, L, T, M,or V; A15 replaced with G, I, L, S, T, M, or V; V16 replaced with A, G,I, L, S, T, or M; R18 replaced with H, or K; E19 replaced with D; T21replaced with A, G, I, L, S, M, or V; V22 replaced with A, G, I, L, S,T, or M; Q23 replaced with N; G25 replaced with A, I, L, S, T, M, or V;S26 replaced with A, G, I, L, T, M, or V; E27 replaced with D; T28replaced with A, G, I, L, S, M, or V; G29 replaced with A, I, L, S, T,M, or V; S31 replaced with A, G, I, L, T, M, or V; E33 replaced with D;W34 replaced with F, or Y; M35 replaced with A, G, I, L, S, T, or V; L36replaced with A, G, I, S, T, M, or V; Q37 replaced with N; H38 replacedwith K, or R; D39 replaced with E; L40 replaced with A, G, I, S, T, M,or V; 141 replaced with A, G, L, S, T, M, or V; G43 replaced with A, I,L, S, T, M, or V; D44 replaced with E; L45 replaced with A, G, I, S, T,M, or V; R46 replaced with H, or K; D47 replaced with E; L48 replacedwith A, G, I, S, T, M, or V; R49 replaced with H, or K; V50 replacedwith A, G, I, L, S, T, or M; E51 replaced with D; V53 replaced with A,G, I, L, S, T, or M; T54 replaced with A, G, I, L, S, M, or V; T55replaced with A, G, I, L, S, M, or V; S56 replaced with A, G, I, L, T,M, or V; V57 replaced with A, G, I, L, S, T, or M; A58 replaced with G,I, L, S, T, M, or V; T59 replaced with A, G, I, L, S, M, or V; G60replaced with A, I, L, S, T, M, or V; D61 replaced with E; Y62 replacedwith F, or W; S63 replaced with A, G, I, L, T, M, or V; 164 replacedwith A, G, L, S, T, M, or V; L65 replaced with A, G, I, S, T, M, or V;M66 replaced with A, G, I, L, S, T, or V; N67 replaced with Q; V68replaced with A, G, I, L, S, T, or M; S69 replaced with A, G, I, L, T,M, or V; W70 replaced with F, or Y; V71 replaced with A, G, I, L, S, T,or M; L72 replaced with A, G, I, S, T, M, or V; R73 replaced with H, orK; A74 replaced with G, I, L, S, T, M, or V; D75 replaced with E; A76replaced with G, I, L, S, T, M, or V; S77 replaced with A, G, I, L, T,M, or V; 178 replaced with A, G, L, S, T, M, or V; R79 replaced with H,or K; L80 replaced with A, G, I, S, T, M, or V; L81 replaced with A, G,I, S, T, M, or V; K82 replaced with H, or R; A83 replaced with G, I, L,S, T, M, or V; T84 replaced with A, G, I, L, S, M, or V; K85 replacedwith H, or R; 186 replaced with A, G, L, S, T, M, or V; V88 replacedwith A, G, I, L, S, T, or M; T89 replaced with A, G, I, L, S, M, or V;G90 replaced with A, I, L, S, T, M, or V; K91 replaced with H, or R; S92replaced with A, G, I, L, T, M, or V; N93 replaced with Q; F94 replacedwith W, or Y; Q95 replaced with N; S96 replaced with A, G, I, L, T, M,or V; Y97 replaced with F, or W; S98 replaced with A, G, I, L, T, M, orV; V100 replaced with A, G, I, L, S, T, or M; R101 replaced with H, orK; N103 replaced with Q; Y104 replaced with F, or W; T105 replaced withA, G, I, L, S, M, or V; E106 replaced with D; A107 replaced with G, I,L, S, T, M, or V; F108 replaced with W, or Y; Q109 replaced with N; T110replaced with A, G, I, L, S, M, or V; Q111 replaced with N; T112replaced with A, G, I, L, S, M, or V; R113 replaced with H, or K; S115replaced with A, G, I, L, T, M, or V; G116 replaced with A, I, L, S, T,M, or V; G117 replaced with A, I, L, S, T, M, or V; K118 replaced withH, or R; W119 replaced with F, or Y; T120 replaced with A, G, I, L, S,M, or V; F121 replaced with W, or Y; S122 replaced with A, G, I, L, T,M, or V; Y123 replaced with F, or W; I124 replaced with A, G, L, S, T,M, or V; G125 replaced with A, I, L, S, T, M, or V; F126 replaced withW, or Y; V128 replaced with A, G, I, L, S, T, or M; E129 replaced withD; L130 replaced with A, G, I, S, T, M, or V; N131 replaced with Q; T132replaced with A, G, I, L, S, M, or V; V133 replaced with A, G, I, L, S,T, or M; Y134 replaced with F, or W; F135 replaced with W, or Y; I136replaced with A, G, L, S, T, M, or V; G137 replaced with A, I, L, S, T,M, or V; A138 replaced with G, I, L, S, T, M, or V; H139 replaced withK, or R; N140 replaced with Q; I141 replaced with A, G, L, S, T, M, orV; N143 replaced with Q; A144 replaced with G, I, L, S, T, M, or V; N145replaced with Q; M146 replaced with A, G, I, L, S, T, or V; N147replaced with Q; E148 replaced with D; D149 replaced with E; G150replaced with A, I, L, S, T, M, or V; S152 replaced with A, G, I, L, T,M, or V; M153 replaced with A, G, I, L, S, T, or V; S154 replaced withA, G, I, L, T, M, or V; V155 replaced with A, G, I, L, S, T, or M; N156replaced with Q; F157 replaced with W, or Y; T158 replaced with A, G, I,L, S, M, or V; S159 replaced with A, G, I, L, T, M, or V; G161 replacedwith A, I, L, S, T, M, or V; L163 replaced with A, G, S, T, M, or V;D164 replaced with E; H165 replaced with K, or R; I166 replaced with A,G, L, S, T, M, or V; M167 replaced with A, G, I, L, S, T, or V; K168replaced with H, or R; Y169 replaced with F, or W; K170 replaced with H,or R; K171 replaced with H, or R; K172 replaced with H, or R; V174replaced with A, G, I, L, S, T, or M; K175 replaced with H, or R; A176replaced with G, I, L, S, T, M, or V; G177 replaced with A, I, L, S, T,M, or V; S178 replaced with A, G, I, L, T, M, or V; L179 replaced withA, G, I, S, T, M, or V; W180 replaced with F, or Y; D181 replaced withE; N183 replaced with Q; I184 replaced with A, G, L, S, T, M, or V; T185replaced with A, G, I, L, S, M, or V; A186 replaced with G, I, L, S, T,M, or V; K188 replaced with H, or R; K189 replaced with H, or R; N190replaced with Q; E191 replaced with D; E192 replaced with D; T193replaced with A, G, I, L, S, M, or V; V194 replaced with A, G, I, L, S,T, or M; E195 replaced with D; V196 replaced with A, G, I, L, S, T, orM; N197 replaced with Q; F198 replaced with W, or Y; T199 replaced withA, G, I, L, S, M, or V; T200 replaced with A, G, I, L, S, M, or V; T201replaced with A, G, I, L, S, M, or V; L203 replaced with A, G, I, S, T,M, or V; G204 replaced with A, I, L, S, T, M, or V; N₂₀₅ replaced withQ; R206 replaced with H, or K; Y207 replaced with F, or W; M208 replacedwith A, G, I, L, S, T, or V; A209 replaced with G, I, L, S, T, M, or V;L210 replaced with A, G, I, S, T, M, or V; I211 replaced with A, G, L,S, T, M, or V; Q212 replaced with N; H213 replaced with K, or R; 5214replaced with A, G, I, L, T, M, or V; T215 replaced with A, G, I, L, S,M, or V; 1216 replaced with A, G, L, S, T, M, or V; I217 replaced withA, G, L, S, T, M, or V; G218 replaced with A, I, L, S, T, M, or V; F219replaced with W, or Y; S220 replaced with A, G, I, L, T, M, or V; Q221replaced with N; V222 replaced with A, G, I, L, S, T, or M; F223replaced with W, or Y; E224 replaced with D; H226 replaced with K, or R;Q227 replaced with N; K228 replaced with H, or R; K229 replaced with H,or R; Q230 replaced with N; T231 replaced with A, G, I, L, S, M, or V;R232 replaced with H, or K; A233 replaced with G, I, L, S, T, M, or V;S234 replaced with A, G, I, L, T, M, or V; V235 replaced with A, G, I,L, S, T, or M; V236 replaced with A, G, I, L, S, T, or M; 1237 replacedwith A, G, L, S, T, M, or V; V239 replaced with A, G, I, L, S, T, or M;T240 replaced with A, G, I, L, S, M, or V; G241 replaced with A, I, L,S, T, M, or V; D242 replaced with E; S243 replaced with A, G, I, L, T,M, or V; E244 replaced with D; G245 replaced with A, I, L, S, T, M, orV; A246 replaced with G, 1, L, S, T, M, or V; T247 replaced with A, G,I, L, S, M, or V; V248 replaced with A, G, I, L, S, T, or M; Q249replaced with N; L250 replaced with A, G, I, S, T, M, or V; T251replaced with A, G, I, L, S, M, or V; Y253 replaced with F, or W; F254replaced with W, or Y; T256 replaced with A, G, I, L, S, M, or V; G258replaced with A, 1, L, S, T, M, or V; S259 replaced with A, G, I, L, T,M, or V; D260 replaced with E; 1262 replaced with A, G, L, S, T, M, orV; R263 replaced with H, or K; H264 replaced with K, or R; K265 replacedwith H, or R; G266 replaced with A, I, L, S, T, M, or V; T267 replacedwith A, G, I, L, S, M, or V; V268 replaced with A, G, I, L, S, T, or M;V269 replaced with A, G, I, L, S, T, or M; L270 replaced with A, G, I,S, T, M, or V; Q273 replaced with N; T274 replaced with A, G, L, S, M,or V; G275 replaced with A, I, L, S, T, M, or V; V276 replaced with A,G, I, L, S, T, or M; F278 replaced with W, or Y; L280 replaced with A,G, I, S, T, M, or V; D281 replaced with E; N282 replaced with Q; N283replaced with Q; K284 replaced with H, or R; S285 replaced with A, G, I,L, T, M, or V; K286 replaced with H, or R; G288 replaced with A, I, L,S, T, M, or V; G289 replaced with A, I, L, S, T, M, or V; W290 replacedwith F, or Y; L291 replaced with A, G, I, S, T, M, or V; L293 replacedwith A, G, I, S, T, M, or V; L294 replaced with A, G, I, S, T, M, or V;L295 replaced with A, G, I, S, T, M, or V; L296 replaced with A, G, I,S, T, M, or V; S297 replaced with A, G, I, L, T, M, or V; L298 replacedwith A, G, I, S, T, M, or V; L299 replaced with A, G, I, S, T, M, or V;V300 replaced with A, G, I, L, S, T, or M; A301 replaced with G, I, L,S, T, M, or V; T302 replaced with A, G, I, L, S, M, or V; W303 replacedwith F, or Y; V304 replaced with A, G, I, L, S, T, or M; L305 replacedwith A, G, I, S, T, M, or V; V306 replaced with A, G, L, S, T, or M;A307 replaced with G, I, L, S, T, M, or V; G308 replaced with A, I, L,S, T, M, or V; 1309 replaced with A, G, L, S, T, M, or V; Y310 replacedwith F, or W; L311 replaced with A, G, I, S, T, M, or V; M312 replacedwith A, G, I, L, S, T, or V; W313 replaced with F, or Y; R314 replacedwith H, or K; H315 replaced with K, or R; E316 replaced with D; R317replaced with H, or K; 1318 replaced with A, G, L, S, T, M, or V; K319replaced with H, or R; K320 replaced with H, or R; T321 replaced with A,G, I, L, S, M, or V; S322 replaced with A, G, I, L, T, M, or V; F323replaced with W, or Y; S324 replaced with A, G, I, L, T, M, or V; T325replaced with A, G, I, L, S, M, or V; T326 replaced with A, G, I, L, S,M, or V; T327 replaced with A, G, I, L, S, M, or V; L328 replaced withA, G, I, S, T, M, or V; L329 replaced with A, G, I, S, T, M, or V; I332replaced with A, G, L, S, T, M, or V; K333 replaced with H, or R; V334replaced with A, G, I, L, S, T, or M; L335 replaced with A, G, I, S, T,M, or V; V336 replaced with A, G, I, L, S, T, or M; V337 replaced withA, G, I, L, S, T, or M; Y338 replaced with F, or W; S340 replaced withA, G, I, L, T, M, or V; E341 replaced with D; 1342 replaced with A, G,L, S, T, M, or V; F344 replaced with W, or Y; H345 replaced with K, orR; H346 replaced with K, or R; T347 replaced with A, G, I, L, S, M, orV; I348 replaced with A, G, L, S, T, M, or V; Y350 replaced with F, orW; F351 replaced with W, or Y; T352 replaced with A, G, I, L, S, M, orV; E353 replaced with D; F354 replaced with W, or Y; L355 replaced withA, G, I, S, T, M, or V; Q356 replaced with N; N357 replaced with Q; H358replaced with K, or R; R360 replaced with H, or K; S361 replaced with A,G, I, L, T, M, or V; E362 replaced with D; V363 replaced with A, G, I,L, S, T, or M; I364 replaced with A, G, L, S, T, M, or V; L365 replacedwith A, G, I, S, T, M, or V; E366 replaced with D; K367 replaced with H,or R; W368 replaced with F, or Y; Q369 replaced with N; K370 replacedwith H, or R; K371 replaced with H, or R; K372 replaced with H, or R;I373 replaced with A, G, L, S, T, M, or V; A374 replaced with G, I, L,S, T, M, or V; E375 replaced with D; M376 replaced with A, G, I, L, S,T, or V; G377 replaced with A, I, L, S, T, M, or V; V379 replaced withA, G, I, L, S, T, or M; Q380 replaced with N; W381 replaced with F, orY; L382 replaced with A, G, I, S, T, M, or V; A383 replaced with G, I,L, S, T, M, or V; T384 replaced with A, G, I, L, S, M, or V; Q385replaced with N; K386 replaced with H, or R; K387 replaced with H, or R;A388 replaced with G, I, L, S, T, M, or V; A389 replaced with G, I, L,S, T, M, or V; D390 replaced with E; K391 replaced with H, or R; V392replaced with A, G, I, L, S, T, or M; V393 replaced with A, G, I, L, S,T, or M; F394 replaced with W, or Y; L395 replaced with A, G, I, S, T,M, or V; L396 replaced with A, G, I, S, T, M, or V; S397 replaced withA, G, I, L, T, M, or V; N398 replaced with Q; D399 replaced with E; V400replaced with A, G, I, L, S, T, or M; N401 replaced with Q; 5402replaced with A, G, I, L, T, M, or V; V403 replaced with A, G, I, L, S,T, or M; D405 replaced with E; G406 replaced with A, I, L, S, T, M, orV; T407 replaced with A, G, I, L, S, M, or V; G409 replaced with A, I,L, S, T, M, or V; K410 replaced with H, or R; S411 replaced with A, G,I, L, T, M, or V; E412 replaced with D; G413 replaced with A, I, L, S,T, M, or V; S414 replaced with A, G, I, L, T, M, or V; 5416 replacedwith A, G, I, L, T, M, or V; E417 replaced with D; N418 replaced with Q;S419 replaced with A, G, I, L, T, M, or V; Q420 replaced with N; D421replaced with E; S422 replaced with A, G, I, L, T, M, or V; S423replaced with A, G, I, L, T, M, or V; and L426 replaced with A, G, I, S,T, M, or V in the amino acid sequence shown in SEQ ID NO:2.

The resulting constructs can be routinely screened for activities orfunctions described throughout the specification and known in the art.Preferably, the resulting constructs have an increased and/or adecreased IL17RLP activity or function, while the remaining IL17RLPactivities or functions are maintained. More preferably, the resultingconstructs have more than one increased and/or decreased IL17RLPactivity or function, while the remaining IL17RLP activities orfunctions are maintained.

Besides conservative amino acid substitution, variants of IL17RLPinclude (i) substitutions with one or more of the non-conserved aminoacid residues, where the substituted amino acid residues may or may notbe one encoded by the genetic code, or (ii) substitution with one ormore of amino acid residues having a substituent group, or (iii) fusionof the mature polypeptide with another compound, such as a compound toincrease the stability and/or solubility of the polypeptide (forexample, polyethylene glycol), or (iv) fusion of the polypeptide withadditional amino acids, such as, for example, an IgG Fc fusion regionpeptide, or leader or secretory sequence, or a sequence facilitatingpurification. Such variant polypeptides are deemed to be within thescope of those skilled in the art from the teachings herein.

For example, IL17RLP polypeptide variants containing amino acidsubstitutions of charged amino acids with other charged or neutral aminoacids may produce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

For example, preferred non-conservative substitutions of HA 7RLPinclude: M1 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S2replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L3 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; V4 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; L5 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; L6 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S7replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L8 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; A9 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; A10 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; L11 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C12replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; R13 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; S14 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A15 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V16 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; P17 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; R18 replaced with D, E, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; E19 replaced with H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; P20 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; T21 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; V22 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; Q23 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,F, W, Y, P, or C; C24 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, or P; G25 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; S26 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E27replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;T28 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G29 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; P30 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S31 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; P32 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E33 replaced with H, K, R, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; W34 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; M35 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; L36 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; Q37 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,F, W, Y, P, or C; H38 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; D39 replaced with H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; L40 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; 141 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P42replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; G43 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D44 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L45replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R46 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D47 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L48 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; R49 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V50 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; E51 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; P52 replaced with D, E, H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, or C; V53 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; T54 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; T55 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S56replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V57 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; A58 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; T59 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; G60 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D61replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;Y62 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;S63 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I64 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L65 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; M66 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; N67 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,F, W, Y, P, or C; V68 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; S69 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W70 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; V71 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L72 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; R73 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; A74 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; D75 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; A76 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; S77 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 178 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; R79 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L80 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; L81 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; K82 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; A83 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;T84 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K85 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 186 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; C87 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V88 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; T89 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; G90 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; K91 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; S92 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N93replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;F94 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;Q95 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; S96 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y97 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S98 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; C99 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V100 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; R101 replaced with D, E, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; C102 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, N, Q, F, W, Y, or P; N103 replaced with D, E, H, K,R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; Y104 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T105 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; E106 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; A107 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; F108 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,T, M, V, P, or C; Q109 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, F, W, Y, P, or C; T110 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; Q111 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,P, or C; T112 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R113replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P114replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; S115 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G116replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G117 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; K118 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; W119 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; T120 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; F121 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; S122 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; Y123 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; I124 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G125replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F126 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; P127 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; V128replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E129 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L130 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; N131 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T132 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; V133 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; Y134 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,T, M, V, P, or C; F135 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,T, M, V, P, or C; I136 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; G137 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A138replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H139 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N140 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; I141 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; P142 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; N143 replaced with D,E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A144 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; N145 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; M146 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; N147 replaced with D, E, H, K, R, A, G, I, L,S, T, M, V, F, W, Y, P, or C; E148 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; D149 replaced with H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; G150 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; P151 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, or C; 5152 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; M153 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;S154 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V155 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; N156 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; F157 replaced with D, E,H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T158 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; 5159 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; P160 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, or C; G161 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; C162 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, or P; L163 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;D164 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; H165 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; I166 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M167replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K168 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Y169 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K170 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K171 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K172 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C173 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V174replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K175 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A176 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; G177 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; S178 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; L179 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W180replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D181replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;P182 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or C; N183 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,P, or C; I184 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T185replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A186 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; C187 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; K188 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K189 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N190 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E191 replaced with H, K,R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E192 replaced with H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T193 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V194 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; E195 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; V196 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; N197 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; F198 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,M, V, P, or C; T199 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;T200 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T201 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; P202 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L203 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; G204 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; N₂O₅ replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, F, W, Y, P, or C; R206 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; Y207 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; M208 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; A209 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L210replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I211 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; Q212 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; H213 replaced with D, E, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S214 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; T215 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; 1216 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;I217 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G218 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; F219 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; 5220 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; Q221 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, F, W, Y, P, or C; V222 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; F223 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,T, M, V, P, or C; E224 replaced with H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, P, or C; P225 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, or C; H226 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; Q227 replaced with D, E, H, K, R, A, G, I, L,S, T, M, V, F, W, Y, P, or C; K228 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; K229 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; Q230 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, F, W, Y, P, or C; T231 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; R232 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,F, W, Y, P, or C; A233 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; S234 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V235replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V236 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; 1237 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; P238 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or C; V239 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; T240 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;G241 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D242 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S243replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E244 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G245 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; A246 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; T247 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; V248 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;Q249 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; L250 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T251replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P252 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; Y253replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F254replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; P255replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; T256 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C257replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; G258 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S259replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D260 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C261 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; 1262replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R263 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H264 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K265 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G266 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; T267 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; V268 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; V269 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L270replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C271 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; P272replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; Q273 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; T274 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G275replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V276 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; P277 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; F278 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; P279 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L280 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; D281 replaced with H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; N282 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; N283 replaced with D, E, H, K,R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K284 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 5285 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; K286 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; P287 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; G288 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; G289 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; W290 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; L291 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P292replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; L293 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L294replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L295 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L296 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; S297 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; L298 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L299replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V300 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; A301 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; T302 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; W303 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; V304 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L305replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V306 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; A307 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; G308 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; 1309 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y310replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L311replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M312 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; W313 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; R314 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; H315 replaced with D, E, A, G, I,L, S, T, M, V, N, Q, F, W, Y, P, or C; E316 replaced with H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R317 replaced with D, E, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 1318 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; K319 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; K320 replaced with D, E, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; T321 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; S322 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;F323 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;S324 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T325 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; T326 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; T327 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L328 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L329 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P330 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P331replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; 1332 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K333replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V334replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L335 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V336 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; V337 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; Y338 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; P339 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,F, W, Y, or C; S340 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;E341 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; I342 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C343replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; F344 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, orC; H345 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; H346 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; T347 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I348replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C349 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; Y350replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; F351replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T352replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E353 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F354 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L355 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; Q356 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; N357 replaced with D, E,H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; H358 replaced with D,E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C359 replaced with D,E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; R360 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 5361 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; E362 replaced with H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V363 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; 1364 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; L365 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; E366 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; K367 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; W368 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; Q369 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,P, or C; K370 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; K371 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; K372 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; 1373 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A374replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E375 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; M376 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; G377 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; P378 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; V379 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; Q380 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, F, W, Y, P, or C; W381 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; L382 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; A383 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T384replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q385 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K386 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K387 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A388 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; A389 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; D390 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; K391 replaced with D, E, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; V392 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; V393 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; F394 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, orC; L395 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L396replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 5397 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; N398 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, F, W, Y, P, or C; D399 replaced with H, K, R, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V400 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; N401 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, F, W, Y, P, or C; S402 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; V403 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; C404 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or P; D405 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; G406 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;T407 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C408 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; G409replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K410 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 5411 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; E412 replaced with H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G413 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; S414 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; P415 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,Q, F, W, Y, or C; 5416 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; E417 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; N418 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,P, or C; S419 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q420replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C;D421 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; S422 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; 5423replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P424 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; C425replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orP; and L426 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C in theamino acid sequence shown in SEQ ID NO:2.

The resulting constructs can be routinely screened for activities orfunctions described throughout the specification and known in the art.Preferably, the resulting constructs have an increased and/or decreasedIL17RLP activity or function, while the remaining IL17RLP activities orfunctions are maintained. More preferably, the resulting constructs havemore than one increased and/or decreased IL17RLP activity or function,while the remaining IL17RLP activities or functions are maintained.

Additionally, more than one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9 and10) can be replaced with the substituted amino acids as described above(either conservative or nonconservative). The substituted amino acidscan occur in the full length, mature, or proprotein form of IL17RLPprotein, as well as the N- and C-terminal deletion mutants, having thegeneral formula n-m, listed above (e.g., n¹-m¹, n¹-m², n¹-m³, n²-m¹,n²-m², n²-m³, n³-m¹, n³-m², and/or n³-m³).

Embodiments of the invention are directed to polypeptides which comprisethe amino acid sequence of an IL17RLP polypeptide described herein, buthaving an amino acid sequence which contains at least one conservativeamino acid substitution, but not more than 50 conservative amino acidsubstitutions, even more preferably, not more than 40 conservative aminoacid substitutions, still more preferably, not more than 30 conservativeamino acid substitutions, and still even more preferably, not more than20 conservative amino acid substitutions, when compared with thefollistatin-3 polynucleotide sequence described herein. Of course, inorder of ever-increasing preference, it is highly preferable for apeptide or polypeptide to have an amino acid sequence which comprisesthe amino acid sequence of an IL17RLP polypeptide, which contains atleast one, but not more than 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1conservative amino acid substitutions.

In further specific embodiments, the number of substitutions, additionsor deletions in the amino acid sequence of FIGS. 1A, 1B, and 1C (SEQ IDNO:2), a polypeptide sequence encoded by the deposited clones, and/orany of the polypeptide fragments described herein is 150, 100, 75, 70,60, 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or250-150, 200-50, 150-50, 100-50, 50-20, 30-20, 20-15, 20-10, 15-10,10-1, 5-10, 1-5, 1-3 or 1-2.

To improve or alter the characteristics of IL17RLP polypeptides, proteinengineering may be employed. Recombinant DNA technology known to thoseskilled in the art can be used to create novel mutant proteins ormuteins including single or multiple amino acid substitutions,deletions, additions or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions.

Thus, the invention also encompasses IL17RLP derivatives and analogsthat have one or more amino acid residues deleted, added, or substitutedto generate IL17RLP polypeptides that are better suited for expression,scale up, etc., in the host cells chosen. For example, cysteine residuescan be deleted or substituted with another amino acid residue in orderto eliminate disulfide bridges, PKC phosphorylation sites, CK2phosphorylation sites, cAMP- and cGMP-dependent protein kinasephosphorylation sites, myristolation, and/or N-linked glycosylationsites can be altered or eliminated to achieve an altered function orexpression pattern of the polypeptide (for example, a mutated N-linkedglycosylation site may alter the expression of a homogeneous productthat is more easily recovered and purified from yeast hosts which areknown to hyperglycosylate N-linked sites). To this end, a variety ofamino acid substitutions at one or both of the first or third amino acidpositions on any one or more of the disulfide bridge cysteines, PKCphosphorylation sites, CK2 phosphorylation sites, cAMP- andcGMP-dependent protein kinase phosphorylation sites, myristolation,and/or glycosylation recognition sequences in the IL17RLP polypeptidesof the invention, and/or an amino acid deletion at the second positionof any one or more such recognition sequences will alter function orexpression or prevent glycosylation of the IL17RLP polypeptide at themodified tripeptide sequence (see, e.g., Miyajima, A., et al., EMBO J.5(6):1193-1197 (1986)).

Amino acids in the IL17RLP protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro proliferative activity.

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids which may produce proteins with highlydesirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic (Pinckard, et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins, et al., Diabetes 36:838-845 (1987); Cleland, et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993)).

Replacement of amino acids can also change the selectivity of thebinding of a ligand to cell surface receptors (for example, Ostade, etal., Nature 361:266-268 (1993)) describes certain mutations resulting inselective binding of TNF-alpha to only one of the two known types of TNFreceptors. Sites that are critical for ligand-receptor binding can alsobe determined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith, et al., J. Mol.Biol. 224:899-904 (1992); de Vos, et al. Science 255:306-312 (1992)).

Since IL17RLP is a homologue of the murine IL-17 receptor protein, tomodulate rather than completely eliminate biological activities ofIL17RLP preferably mutations are made in sequences encoding amino acidsin the IL17RLP conserved extracellular domain, i.e., in positions I-271of SEQ ID NO:2, more preferably in residues within this region which arenot conserved in the murine IL-17 receptor protein. Also forming part ofthe present invention are isolated polynucleotides comprising nucleicacid sequences which encode the above IL17RLP mutants.

Amino acid regions of the IL17RLP sequence shown in SEQ ID NO:2 whichare highly conserved when compared to the murine IL-17R polypeptidesequence shown as SEQ ID NO:3 (see FIG. 2) are attractive regions fortargeted mutagenesis of the IL17RLP polypeptides of the invention. Infact, a number of conserved regions or domains have been set forth inFIGS. 1A, 1B, and 1C (labeled as Domains I-VIII). These domains are asfollows: Domain I (i.e., Val-49 through Leu-62 of SEQ ID NO:2 (Val-68through Leu-81 of FIGS. 1A, 1B, and 1C)); Domain II (Cys-154 throughThr-166 of SEQ ID NO:2 (i.e., Cys-173 through Thr-185 of FIGS. 1A, 1B,and 1C)); Domain III (Gln-202 through Gln-208 of SEQ ID NO:2 (i.e.,Gln-221 through Gln-227 of FIGS. 1A, 1B, and 1C)); Domain IV (Asp-241through Val-249 of SEQ ID NO:2 (i.e., Asp-260 through Val-268 of FIGS.1A, 1B, and 1C)); Domain V (Thr-255 through Leu-261 of SEQ ID NO:2(i.e., Thr-274 through Leu-280 of FIGS. 1A, 1B, and 1C)); Domain VI(Leu-310 through Tyr-319 of SEQ ID NO:2 (i.e., Leu-329 through Tyr-338of FIGS. 1A, 1B, and 1C)); Domain VII (Cys-340 through Leu-346 of SEQ IDNO:2 (i.e., Cys-359 through Leu-365 of FIGS. 1A, 1B, and 1C)); andDomain VIII (Ile-354 through Gly-358 of SEQ ID NO:2 (i.e., Ile-373through Gly-377 of FIGS. 1A, 1B, and 1C)).

In another embodiment of the invention, seven cysteine residues ofIL17RLP are conserved with respect to the murine IL-17R polypeptidesequence shown in SEQ ID NO:3. Cysteine residues tend to play animportant role in the structural conformation, and thus, the function ofa polypeptide. As such, the seven conserved cysteine residues are alsoattractive residues for targeted mutagenesis of the IL17RLP polypeptidesof the invention. The seven highly conserved cysteine residues of theIL17RLP shown in SEQ ID NO:2 of the present invention are as follows:Cys-5, Cys-80, Cys-143, Cys-154, Cys-238, Cys-242, and Cys-340 of SEQ IDNO:2 (which correspond exactly to Cys-24, Cys-99, Cys-162, Cys-173,Cys-257, Cys-261, and Cys-359 of FIGS. 1A, 1B, and 1C).

Thus, a polynucleotide encoding a polypeptide comprising, oralternatively consisting of, a member selected from the group consistingof amino acid residues Cys-5 to Cys-340, Cys-80 to Cys-340, Cys-143 toCys-340, Cys-154 to Cys-340, Cys-238 to Cys-340, Cys-242 to Cys-340,Cys-5 to Cys-242, Cys-80 to Cys-242, Cys-143 to Cys-242, Cys-154 toCys-242, Cys-238 to Cys-242, Cys-5 to Cys-238, Cys-80 to Cys-238,Cys-143 to Cys-238, Cys-154 to Cys-238, Cys-5 to Cys-154, Cys-80 toCys-154, Cys-143 to Cys-154, Cys-5 to Cys-143, Cys-80 to Cys-143, andCys-5 to Cys-80 of the IL17RLP amino acid sequence shown as SEQ ID NO:2is a preferred polynucleotide fragment of the present invention.Polypeptides encoded by these polynucleotides are also encompassed bythe invention. The present invention is also directed to nucleic acidmolecules comprising, or alternatively, consisting of, a polynucleotidesequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100%identical to the polynucleotide sequence encoding the IL17RLPpolypeptides described above. The present invention also encompasses theabove polynucleotide sequences fused to a heterologous polynucleotidesequence. Polypeptides encoded by these nucleic acids and/orpolynucleotide sequences are also encompassed by the invention, as arepolypeptides comprising, or alternatively consisting of, an amino acidsequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence described above, andpolynucleotides that encode such polypeptides.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of the IL17RLP polypeptide can besubstantially purified by the one-step method described by Smith andJohnson (Gene 67:31-40 (1988)). Polypeptides of the invention also canbe purified from natural or recombinant sources using anti-IL17RLPantibodies of the invention in methods which are well known in the artof protein purification.

The invention further provides an isolated IL17RLP polypeptidecomprising, or alternatively consisting of, an amino acid sequenceselected from the group consisting of: (a) the amino acid sequence ofthe full-length IL17RLP polypeptide having the complete amino acidsequence shown in SEQ ID NO:2 (i.e., positions −19 to 407 of SEQ IDNO:2); (b) the amino acid sequence of the full-length IL17RLPpolypeptide having the complete amino acid sequence shown in SEQ ID NO:2excepting the N-terminal methionine (i.e., positions −18 to 407 of SEQID NO:2); (c) the amino acid sequence of the mature IL17RLP polypeptidehaving the complete amino acid sequence shown in SEQ ID NO:2 (i.e.,positions 1 to 407 of SEQ ID NO:2); (d) the amino acid sequence of thepredicted extracellular domain of the IL17RLP polypeptide having thecomplete amino acid sequence shown in SEQ ID NO:2 (i.e., positions 1 to271 of SEQ ID NO:2); (e) the amino acid sequence of a soluble IL17RLPpolypeptide having the predicted extracellular and intracellulardomains, but lacking the predicted transmembrane domain; (f) thecomplete amino acid sequence encoded by the human cDNA contained in theATCC™ Deposit No. 209198; (g) the complete amino acid sequence exceptingthe N-terminal methionine encoded by the human cDNA contained in theATCC™ Deposit No. 209198; (h) the complete amino acid sequence of themature IL17RLP encoded by the human cDNA contained in the ATCC™ DepositNo. 209198, and; (i) the complete amino acid sequence of theextracellular domain of the IL17RLP encoded by the human cDNA containedin the ATCC™ Deposit No. 209198. The polypeptides of the presentinvention also include polypeptides having an amino acid sequence atleast 80% identical, more preferably at least 90% identical, and stillmore preferably 95%, 96%, 97%, 98%, 99%, or 100% identical to thosedescribed in (a), (b), (c), (d), (e), (f), (g), (h) or (i) above, aswell as polypeptides having an amino acid sequence with at least 90%similarity, and more preferably at least 95% similarity, to those above.

Further polypeptides of the present invention include polypeptides whichhave at least 90% similarity, more preferably at least 95% similarity,and still more preferably at least 96%, 97%, 98%, 99% or 100% similarityto those described above. The polypeptides of the invention alsocomprise those which are at least 80% identical, more preferably atleast 90% or 95% identical, still more preferably at least 96%, 97%,98%, 99% or 100% identical to the polypeptide encoded by the depositedcDNA or to the polypeptide of SEQ ID NO:2, and also include portions ofsuch polypeptides with at least 25 amino acids, at least 30 amino acids,at least 35 amino acids, at least 40 amino acids, at least 45 aminoacids, and more preferably at least 50 amino acids, at least 55 aminoacids, at least 60 amino acids, at least 65 amino acids, at least 70amino acids, at least 75 amino acids, at least 80 amino acids, at least85 amino acids, at least 90 amino acids, at least 95 amino acids, and atleast 100 amino acids.

By “% similarity” for two polypeptides is intended a similarity scoreproduced by comparing the amino acid sequences of the two polypeptidesusing the Bestfit program (Wisconsin Sequence Analysis Package, Version8 for Unix, Genetics Computer Group, University Research Park, 575Science Drive, Madison, Wis. 53711) and the default settings fordetermining similarity. Bestfit uses the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) tofind the best segment of similarity between two sequences.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a IL17RLPpolypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the IL17RLP polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% identical to, forinstance, the amino acid sequence shown in FIGS. 1A, 1B, and 1C (SEQ IDNO:2), the amino acid sequence encoded by deposited cDNA clone HAPOR40,or fragments thereof, can be determined conventionally using knowncomputer programs such the Bestfit program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711). When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference amino acid sequence and that gaps in homology ofup to 5% of the total number of amino acid residues in the referencesequence are allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter. According to this embodiment, if the subject sequence isshorter than the query sequence due to N- or C-terminal deletions, notbecause of internal deletions, a manual correction is made to theresults to take into consideration the fact that the FASTDB program doesnot account for N- and C-terminal truncations of the subject sequencewhen calculating global percent identity. For subject sequencestruncated at the N- and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residues ofthe query sequence that are N- and C-terminal of the subject sequence,which are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. A determination ofwhether a residue is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thisfinal percent identity score is what is used for the purposes of thisembodiment. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence. For example, a 90 aminoacid residue subject sequence is aligned with a 100 residue querysequence to determine percent identity. The deletion occurs at theN-terminus of the subject sequence and therefore, the FASTDB alignmentdoes not show a matching/alignment of the first 10 residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N- and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.In another example, a 90 residue subject sequence is compared with a 100residue query sequence. This time the deletions are internal deletionsso there are no residues at the N- or C-termini of the subject sequencewhich are not matched/aligned with the query. In this case the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N- and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected for. Noother manual corrections are made for the purposes of this embodiment.

The present application is also directed to proteins containingpolypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or100% identical to the IL17RLP polypeptide sequence set forth herein asn¹-m¹, n²-m², and/or n³-m³. In preferred embodiments, the application isdirected to proteins comprising, or alternatively consisting of,polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or100% identical to polypeptides having the amino acid sequence of thespecific IL17RLP N- and C-terminal deletions recited herein.Polynucleotides encoding these polypeptides are also encompassed by theinvention. The present invention also encompasses the above polypeptidesequences fused to a heterologous polypeptide sequence. Polynucleotidesencoding these amino acid sequences are also encompassed by theinvention.

The invention also encompasses fusion proteins in which the full-lengthIL17RLP polypeptide or fragment, variant, derivative, or analog thereofis fused or joined to an unrelated protein. These fusion proteins can beroutinely designed on the basis of the IL17RLP nucleotide andpolypeptide sequences disclosed herein. For example, as one of skill inthe art will appreciate, IL17RLP polypeptides and fragments (includingepitope-bearing fragments) thereof described herein can be combined withparts of the constant domain of immunoglobulins (IgG), resulting inchimeric (fusion) polypeptides. These fusion proteins facilitatepurification and show an increased half-life in vivo. This has beenshown, e.g., for chimeric proteins consisting of the first two domainsof the human CD4-polypeptide and various domains of the constant regionsof the heavy or light chains of mammalian immunoglobulins (EP A 394,827;Traunecker, et al., Nature 331:84-86 (1988)). Fusion proteins that havea disulfide-linked dimeric structure due to the IgG part can also bemore efficient in binding and neutralizing other molecules than themonomeric IL17RLP polypeptide or polypeptide fragments alone(Fountoulakis, et al., J. Biochem. 270:3958-3964 (1995)). Examples ofIL17RLP fusion proteins that are encompassed by the invention include,but are not limited to, fusion of the IL17RLP polypeptide sequences toany amino acid sequence that allows the fusion proteins to be displayedon the cell surface (e.g. the IgG Fc domain); or fusions to an enzyme,fluorescent protein, or luminescent protein which provides a markerfunction.

As described in detail below, the polypeptides of the present inventioncan also be used to raise polyclonal and monoclonal antibodies, whichare useful in assays for detecting IL17RLP protein expression asdescribed below or as agonists and antagonists capable of enhancing orinhibiting IL17RLP protein function. Further, such polypeptides can beused in the yeast two-hybrid system to “capture” IL17RLP protein bindingproteins which are also candidate agonists and antagonists according tothe present invention. The yeast two hybrid system is described byFields and Song (Nature 340:245-246 (1989)).

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. On the other hand, a region of aprotein molecule to which an antibody can bind is defined as an“antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes (see, forinstance, Geysen, et al., Proc. Natl. Acad. Sci. USA 81:3998-4002(1983)).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein (see, for instance, Sutcliffe, J. G., et al., Science219:660-666 (1983)). Peptides capable of eliciting protein-reactive seraare frequently represented in the primary sequence of a protein, can becharacterized by a set of simple chemical rules, and are confinedneither to immunodominant regions of intact proteins (i.e., immunogenicepitopes) nor to the amino or carboxyl terminals. Antigenicepitope-bearing peptides and polypeptides of the invention are thereforeuseful to raise antibodies, including monoclonal antibodies, that bindspecifically to a polypeptide of the invention (see, for instance,Wilson, et al., Cell 37:767-778 (1984)).

Antigenic epitope-bearing peptides and polypeptides of the inventionpreferably contain a sequence of at least seven, more preferably atleast nine and most preferably between about 15 to about 30 amino acidscontained within the amino acid sequence of a polypeptide of theinvention. Non-limiting examples of antigenic polypeptides or peptidesthat can be used to generate IL17RLP-specific antibodies include: apolypeptide comprising, or alternatively consisting of, amino acidresidues from about Ser-14 to about Val-22 in SEQ ID NO:2, a polypeptidecomprising, or alternatively consisting of, amino acid residues fromabout Cys-24 to about Pro-32 in SEQ ID NO:2, a polypeptide comprising,or alternatively consisting of, amino acid residues from about Ile-41 toabout Arg-49 in SEQ ID NO:2, a polypeptide comprising, or alternativelyconsisting of, amino acid residues from about Thr-89 to about Val-97 inSEQ ID NO:2, a polypeptide comprising, or alternatively consisting of,amino acid residues from about Thr-110 to about Lys-118 in SEQ ID NO:2,a polypeptide comprising, or alternatively consisting of, amino acidresidues from about Ala-144 to about Ser-152 in SEQ ID NO:2, apolypeptide comprising, or alternatively consisting of, amino acidresidues from about Thr-240 to about Val-248 in SEQ ID NO:2, apolypeptide comprising, or alternatively consisting of, amino acidresidues from about Gly-258 to about Thr-267 in SEQ ID NO:2, apolypeptide comprising, or alternatively consisting of, amino acidresidues from about Leu-280 to about Gly-288 in SEQ ID NO:2, apolypeptide comprising, or alternatively consisting of, amino acidresidues from about Cys-404 to about Glu-412 in SEQ ID NO:2, apolypeptide comprising, or alternatively consisting of, amino acidresidues from about Pro-415 to about Ser-423 in SEQ ID NO:2, apolypeptide comprising, or alternatively consisting of, amino acidresidues from about Gly-409 to about Glu-417 in SEQ ID NO:2, and apolypeptide comprising, or alternatively consisting of, amino acidresidues from about Cys-404 to about Leu-426 in FIGS. 1A, 1B, and 1C(which is identical to the sequence shown in SEQ ID NO:2 with theexception of the numbering scheme as detailed above). These polypeptidefragments have been determined to bear antigenic epitopes of the IL17RLPprotein by the analysis of the Jameson-Wolf antigenic index, as shown inFIG. 3, above.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means (see, for example, Houghten, R. A.,et al., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985); and U.S. Pat.No. 4,631,211 to Houghten, et al., (1986)).

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art (see, forinstance, Sutcliffe, et al., supra; Wilson, et al., supra; Chow, M, etal., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J., et al.,J. Gen. Virol. 66:2347-2354 (1985)). Immunogenic epitope-bearingpeptides of the invention, i.e., those parts of a protein that elicit anantibody response when the whole protein is the immunogen, areidentified according to methods known in the art (see, for instance,Geysen, et al., supra). Further still, U.S. Pat. No. 5,194,392, issuedto Geysen, describes a general method of detecting or determining thesequence of monomers (amino acids or other compounds) which is atopological equivalent of the epitope (i.e., a “mimotope”) which iscomplementary to a particular paratope (antigen binding site) of anantibody of interest. More generally, U.S. Pat. No. 4,433,092, issued toGeysen, describes a method of detecting or determining a sequence ofmonomers which is a topographical equivalent of a ligand which iscomplementary to the ligand binding site of a particular receptor ofinterest. Similarly, U.S. Pat. No. 5,480,971, issued to Houghten andcolleagues, on Peralkylated Oligopeptide Mixtures discloses linearC1-C7-alkyl peralkylated oligopeptides and sets and libraries of suchpeptides, as well as methods for using such oligopeptide sets andlibraries for determining the sequence of a peralkylated oligopeptidethat preferentially binds to an acceptor molecule of interest. Thus,non-peptide analogs of the epitope-bearing peptides of the inventionalso can be made routinely by these methods.

As one of skill in the art will appreciate, IL17RLP polypeptides of thepresent invention and the epitope-bearing fragments thereof describedabove can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EP A 394,827; Traunecker, et al., Nature 331:84-86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric IL17RLP protein orprotein fragment alone (Fountoulakis, et al., J. Biochem. 270:3958-3964(1995)).

The techniques of gene-shuffling, motif-shuffling, exon-shuffling,and/or codon-shuffling (collectively referred to as “DNA shuffling”) maybe employed to modulate the activities of IL17RLP thereby effectivelygenerating agonists and antagonists of IL17RLP. See generally, U.S. Pat.Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, andPatten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997);Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., etal., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R.Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference). In one embodiment,alteration of IL17RLP polynucleotides and corresponding polypeptides maybe achieved by DNA shuffling. DNA shuffling involves the assembly of twoor more DNA segments into a desired IL17RLP molecule by homologous, orsite-specific, recombination. In another embodiment, IL17RLPpolynucleotides and corresponding polypeptides may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of IL17RLP may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules. In preferred embodiments, the heterologousmolecule is the IL-17 receptor.

In further preferred embodiments, IL17RLP polynucleotides of theinvention are fused to a polynucleotide encoding a “FLAG” polypeptide.Thus, an IL17RLP-FLAG fusion protein is encompassed by the presentinvention. The FLAG antigenic polypeptide may be fused to an IL17RLPpolypeptide of the invention at either or both the amino or the carboxyterminus. In preferred embodiments, an IL17RLP-FLAG fusion protein isexpressed from a pFLAG-CMV-5a or a pFLAG-CMV-1 expression vector(available from SIGMA™, St. Louis, Mo., USA). See, Andersson, S., etal., J. Biol. Chem. 264:8222-29 (1989); Thomsen, D. R., et al., Proc.Natl. Acad. Sci. USA, 81:659-63 (1984); and Kozak, M., Nature 308:241(1984) (each of which is hereby incorporated by reference). In furtherpreferred embodiments, an IL17RLP-FLAG fusion protein is detectable byanti-FLAG monoclonal antibodies (also available from SIGMA™).

The functional activity of IL17RLP polypeptides, and fragments, variantsderivatives, and analogs thereof, can be assayed by various methods.

For example, in one embodiment where one is assaying for the ability tobind or compete with full-length IL17RLP polypeptide for binding to ananti-IL17RLP antibody, various immunoassays known in the art can beused, including but not limited to, competitive and non-competitiveassay systems using techniques such as radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays, immunoradiometricassays, gel diffusion precipitation reactions, immunodiffusion assays,in situ immunoassays (using colloidal gold, enzyme or radioisotopelabels, for example), western blots, precipitation reactions,agglutination assays (e.g., gel agglutination assays, hemagglutinationassays), complement fixation assays, immunofluorescence assays, proteinA assays, and immunoelectrophoresis assays, etc. In one embodiment,antibody binding is detected by detecting a label on the primaryantibody. In another embodiment, the primary antibody is detected bydetecting binding of a secondary antibody or reagent to the primaryantibody. In a further embodiment, the secondary antibody is labeled.Many means are known in the art for detecting binding in an immunoassayand are within the scope of the present invention.

In another embodiment, where an IL17RLP ligand is identified (e.g.IL-20), or the ability of a polypeptide fragment, variant or derivativeof the invention to multimerize is being evaluated, binding can beassayed, e.g., by means well-known in the art, such as, for example,reducing and non-reducing gel chromatography, protein affinitychromatography, and affinity blotting. See generally, Phizicky, E., etal., 1995, Microbiol. Rev. 59:94-123. In another embodiment,physiological correlates of IL17RLP binding to its substrates (signaltransduction) can be assayed.

In addition, assays described herein (see Examples 5-8 and otherwiseknown in the art may routinely be applied to measure the ability ofIL17RLP polypeptides and fragments, variants derivatives and analogsthereof to elicit IL17RLP related biological activity (e.g., to act asan attractant for neutrophils in vitro or in vivo).

Other methods will be known to the skilled artisan and are within thescope of the invention.

The invention further provides for the proteins containing, oralternatively comprising, or alternatively consisting of, polypeptidesequences encoded by the polynucleotides of the invention.

The IL17RLP proteins, or fragments thereof, of the invention may be inmonomers or multimers (i.e., dimers, trimers, tetramers, and highermultimers). Accordingly, the present invention relates to monomers andmultimers of the IL17RLP proteins of the invention, their preparation,and compositions (preferably, pharmaceutical compositions) containingthem. In specific embodiments, the polypeptides of the invention aremonomers, dimers, trimers or tetramers. In additional embodiments, themultimers of the invention are at least dimers, at least trimers, or atleast tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlyIL17RLP proteins of the invention (including IL17RLP fragments,variants, and fusion proteins, as described herein). These homomers maycontain IL17RLP proteins having identical or different polypeptidesequences. In a specific embodiment, a homomer of the invention is amultimer containing only IL17RLP proteins having an identicalpolypeptide sequence. In another specific embodiment, a homomer of theinvention is a multimer containing IL17RLP proteins having differentpolypeptide sequences. In specific embodiments, the multimer of theinvention is a homodimer (e.g., containing IL17RLP proteins havingidentical or different polypeptide sequences) or a homotrimer (e.g.,containing IL17RLP proteins having identical or different polypeptidesequences). In additional embodiments, the homomeric multimer of theinvention is at least a homodimer, at least a homotrimer, or at least ahomotetramer.

As used herein, the term heteromer refers to a multimer containingheterologous proteins (i.e., proteins containing only polypeptidesequences that do not correspond to a polypeptide sequences encoded bythe IL17RLP gene) in addition to the IL17RLP proteins of the invention.In a specific embodiment, the multimer of the invention is aheterodimer, a heterotrimer, or a heterotetramer. In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when proteins of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when proteins of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the IL17RLP proteins of the invention.Such covalent associations may involve one or more amino acid residuescontained in the polypeptide sequence of the polypeptide sequencerecited in SEQ ID NO:2 and contained in the polypeptide encoded by thecDNA clone contained in ATCC™ Deposit No. 209198. In one instance, thecovalent associations are cross-linking between cysteine residueslocated within the polypeptide sequences of the proteins which interactin the native (i.e., naturally occurring) polypeptide. In anotherinstance, the covalent associations are the consequence of chemical orrecombinant manipulation. Alternatively, such covalent associations mayinvolve one or more amino acid residues contained in the heterologouspolypeptide sequence in an IL17RLP fusion protein. In one example,covalent associations are between the heterologous sequence contained ina fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925).In a specific example, the covalent associations are between theheterologous sequence contained in an IL17RLP-Fc fusion protein of theinvention (as described herein).

In another embodiment, the IL17RLP polypeptides of the present inventionand the epitope-bearing fragments thereof are fused with a heterologousantigen (e.g., polypeptide, carbohydrate, phospholipid, or nucleicacid).

In specific embodiments, the heterologous antigen is an immunogen. In amore specific embodiment, the heterologous antigen is the gp120 proteinof HIV, or a fragment thereof. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, proteins desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the polypeptidesequence of the proteins desired to be contained in the multimer (see,e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by referencein its entirety). Further, proteins of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide sequence of the protein and techniquesknown in the art may be applied to generate multimers containing one ormore of these modified proteins (see, e.g., U.S. Pat. No. 5,478,925,which is herein incorporated by reference in its entirety).Additionally, techniques known in the art may be applied to generateliposomes containing the protein components desired to be contained inthe multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, proteinscontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain and which can beincorporated by membrane reconstitution techniques into liposomes (see,e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by referencein its entirety).

In addition, proteins of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., N.Y., andHunkapiller, M., et al., Nature 310:105-111 (1984)). For example, apeptide corresponding to a fragment of the IL17RLP polypeptides of theinvention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into the IL17RLPpolypeptide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

Non-naturally occurring variants may be produced using art-knownmutagenesis techniques, which include, but are not limited tooligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis,site directed mutagenesis (see, e.g., Carter et al., Nucl. Acids Res.13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10:6487 (1982)),cassette mutagenesis (see, e.g., Wells et al., Gene 34:315 (1985)),restriction selection mutagenesis (see, e.g., Wells et al., Philos.Trans. R. Soc. London SerA 317:415 (1986)).

The invention additionally, encompasses IL17RLP polypeptides which aredifferentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques, includingbut not limited to, specific chemical cleavage by cyanogen bromide,trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation,formylation, oxidation, reduction, metabolic synthesis in the presenceof tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofIL17RLP which may provide additional advantages such as increasedsolubility, stability and circulating time of the polypeptide, ordecreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemicalmoieties for derivitization may be selected from water soluble polymerssuch as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. The polypeptides may be modified at random positions within themolecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to a proteins via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) of the protein or to morethan one type of amino acid residue (e.g., lysine, histidine, asparticacid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the invention may beaccomplished by any number of means. For example, polyethylene glycolmay be attached to the protein either directly or by an interveninglinker. Linkerless systems for attaching polyethylene glycol to proteinsare described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998);U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO98/32466, the disclosures of each of which are incorporated herein byreference.

One system for attaching polyethylene glycol directly to amino acidresidues of proteins without an intervening linker employs tresylatedMPEG, which is produced by the modification of monmethoxy polyethyleneglycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Upon reaction ofprotein with tresylated MPEG, polyethylene glycol is directly attachedto amine groups of the protein. Thus, the invention includesprotein-polyethylene glycol conjugates produced by reacting proteins ofthe invention with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number ofdifferent intervening linkers. For example, U.S. Pat. No. 5,612,460, theentire disclosure of which is incorporated herein by reference,discloses urethane linkers for connecting polyethylene glycol toproteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins are describedin WO 98/32466, the entire disclosure of which is incorporated herein byreference. Pegylated protein products produced using the reactionchemistries set out herein are included within the scope of theinvention.

The number of polyethylene glycol moieties attached to each protein ofthe invention (i.e., the degree of substitution) may also vary. Forexample, the pegylated proteins of the invention may be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado of al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

The proteins of the invention can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep,cows and non-human primates, e.g., baboons, monkeys, and chimpanzees maybe used to generate transgenic animals. In a specific embodiment,techniques described herein or otherwise known in the art, are used toexpress polypeptides of the invention in humans, as part of a genetherapy protocol.

Any technique known in the art may be used to introduce the transgene(i.e., nucleic acids of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, Mol. Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety. Further, the contents of each of the documentsrecited in this paragraph is herein incorporated by reference in itsentirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)), each ofwhich is herein incorporated by reference in its entirety).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric animals. The transgene may be integrated as a single transgeneor as multiple copies such as in concatamers, e.g., head-to-head tandemsor head-to-tail tandems. The transgene may also be selectivelyintroduced into and activated in a particular cell type by following,for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl.Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences requiredfor such a cell-type specific activation will depend upon the particularcell type of interest, and will be apparent to those of skill in theart. When it is desired that the polynucleotide transgene be integratedinto the chromosomal site of the endogenous gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous geneare designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous gene. Thetransgene may also be selectively introduced into a particular celltype, thus inactivating the endogenous gene in only that cell type, byfollowing, for example, the teaching of Gu et al. (Gu et al., Science265:103-106 (1994)). The regulatory sequences required for such acell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art. Thecontents of each of the documents recited in this paragraph is hereinincorporated by reference in its entirety.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic and “knock-out” animals of the invention have uses whichinclude, but are not limited to, animal model systems useful inelaborating the biological function of IL17RLP polypeptides, studyingconditions and/or disorders associated with aberrant IL17RLP expression,and in screening for compounds effective in ameliorating such conditionsand/or disorders.

In further embodiments of the invention, cells that are geneticallyengineered to express the proteins of the invention, or alternatively,that are genetically engineered not to express the proteins of theinvention (e.g., knockouts) are administered to a patient in vivo. Suchcells may be obtained from the patient (i.e., animal, including human)or an MHC compatible donor and can include, but are not limited tofibroblasts, bone marrow cells, blood cells (e.g., lymphocytes),adipocytes, muscle cells, endothelial cells, etc. The cells aregenetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally. Alternatively, the cells can be incorporated into amatrix and implanted in the body, e.g., genetically engineeredfibroblasts can be implanted as part of a skin graft; geneticallyengineered endothelial cells can be implanted as part of a lymphatic orvascular graft. (See, for example, Anderson et al. U.S. Pat. No.5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959, each of whichis incorporated by reference herein in its entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Antibodies

The present invention further relates to antibodies and T-cell antigenreceptors (TCR) which immunospecifically bind a polypeptide, preferablyan epitope, of the present invention (as determined by immunoassays wellknown in the art for assaying specific antibody-antigen binding).Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. The term“antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)₂, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine, donkey, shiprabbit, goat, guinea pig, camel, horse, or chicken. As used herein,“human” antibodies include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulin and that do not express endogenous immunoglobulins,as described infra and, for example in, U.S. Pat. No. 5,939,598 byKucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention that they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies thatspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. Antibodies that do not bind polypeptides with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a polypeptide of the present invention are also included inthe present invention. Further included in the present invention areantibodies that bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻²M, 10⁻²M, 5×10⁻³M, 10⁻³M, 5×10⁻⁴M, 10⁻⁴M, 5×10⁻⁵M, 10⁻⁵M, 5×10⁻⁶M,10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M,5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M,5×10⁻¹⁵M, and 10⁻¹⁵M.

The invention also provides antibodies that competitively inhibit thebinding of a monoclonal antibody to a polypeptide of the invention,preferably the polypeptide of SEQ ID NO:2. Competitive inhibition can bedetermined by any method known in the art, for example, using thecompetitive binding assays described herein. In preferred embodiments,the antibody competitively inhibits the binding of a monoclonal antibodyof the invention by at least 90%, at least 80%, at least 70%, at least60%, or at least 50% to the polypeptide of SEQ ID NO:2.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. The invention features both receptor-specific antibodies andligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orits substrate by immunoprecipitation followed by western blot analysis(for example, as described supra). In specific embodiments, antibodiesare provided that inhibit ligand or receptor activity by at least 90%,at least 80%, at least 70%, at least 60%, or at least 50% of theactivity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex. Also included arereceptor-specific antibodies that do not specifically recognize theunbound receptor or the unbound ligand. Likewise, included in theinvention are neutralizing antibodies which bind the ligand and preventbinding of the ligand to the receptor, as well as antibodies which bindthe ligand, thereby preventing receptor activation, but do not preventthe ligand from binding the receptor. Further included in the inventionare antibodies which activate the receptor. These antibodies may act asreceptor agonists, i.e., potentiate or activate either all or a subsetof the biological activities of the ligand-mediated receptor activation.The antibodies may be specified as agonists, antagonists or inverseagonists for biological activities comprising the specific biologicalactivities of the peptides of the invention disclosed herein. The aboveantibody agonists can be made using methods known in the art. See, e.g.,PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood92(6):1981-1988 (1998); Chen, et al., Cancer Res. 58(16):3668-3678(1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al.,Cancer Res. 58(15):3209-3214 (1998); Yoon, et al., J. Immunol.160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247(1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997);Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol.Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762(1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al.,Cytokine 8(1):14-20 (1996) (which are all incorporated by referenceherein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, or toxins. See, e.g., PCT publicationsWO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP396,387.

The antibodies of the invention include derivatives that are modified,i.e, by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interestcan be produced by various procedures well known in the art. Forexample, a polypeptide of the invention can be administered to varioushost animals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well-known in the art and arediscussed in detail in Example 10. Briefly, mice can be immunized with apolypeptide of the invention or a cell expressing such peptide. Once animmune response is detected, e.g., antibodies specific for the antigenare detected in the mouse serum, the mouse spleen is harvested andsplenocytes isolated. The splenocytes are then fused by well-knowntechniques to any suitable myeloma cells, for example cells from cellline SP20 available from the ATCC™. Hybridomas are selected and clonedby limited dilution. The hybridoma clones are then assayed by methodsknown in the art for cells that secrete antibodies capable of binding apolypeptide of the invention. Ascites fluid, which generally containshigh levels of antibodies, can be generated by immunizing mice withpositive hybridoma clones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporatedherein by reference in their entireties. Humanized antibodies areantibody molecules from non-human species antibody that binds thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and framework regions from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment, diagnosis, and/or detection of human patients. Humanantibodies can be made by a variety of methods known in the artincluding phage display methods described above using antibody librariesderived from human immunoglobulin sequences. See also, U.S. Pat. Nos.4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433,WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741;each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat.Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; and 5,939,598, which are incorporated by reference herein intheir entirety. In addition, companies such as ABGENIX™, Inc. (Freemont,Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

The term “bind(ing) of a polypeptide of the invention to a ligand”includes, but is not limited to, the binding of a ligand polypeptide ofthe present invention to a receptor, the binding of a receptorpolypeptide of the present invention to a ligand; the binding of anantibody of the present invention to an antigen or epitope; the bindingof an antigen or epitope of the present invention to an antibody; thebinding of an antibody of the present invention to an anti-idiotypicantibody; the binding of an anti-idiotypic antibody of the presentinvention to a ligand; the biding of an anti-idiotypic antibody of thepresent invention to a receptor; the binding of an anti-anti-idiotypicantibody of the present invention to a ligand, receptor or antibody,etc.

As another example, antibodies which bind to and competitively activatethe polypeptide of the invention or its ligand can be used to generateanti-idiotypic antibodies that mimic the polypeptide binding domainand/or activation domain and, as a consequence, bind to and activate thepolypeptide and/or its ligand. Such activating anti-idiotypes or Fabfragments of such anti-idiotypes can be used in therapeutic regimens toactivate polypeptide ligand. For example, such anti-idiotypic antibodiescan be used to bind a polypeptide of the invention to thereby activateits biological activity and/or bind a ligand/receptor of the polypeptideof the invention to thereby activate its biological activity.

Polynucleotides Encoding Antibodies.

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:2 or SEQ ID NO:18.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligation of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be obtained from a suitable source (e.g., an antibodycDNA library, or a cDNA library generated from, or nucleic acid,preferably poly A+ RNA, isolated from, any tissue or cells expressingthe antibody, such as hybridoma cells selected to express an antibody ofthe invention) by PCR amplification using synthetic primers hybridizableto the 3′ and 5′ ends of the sequence or by cloning using anoligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions. In a specific embodiment, the amino acid sequence ofthe heavy and/or light chain variable domains may be inspected toidentify the sequences of the complementarity determining regions (CDRs)by methods that are well know in the art, e.g., by comparison to knownamino acid sequences of other heavy and light chain variable regions todetermine the regions of sequence hypervariability. Using routinerecombinant DNA techniques, one or more of the CDRs may be insertedwithin framework regions, e.g., into human framework regions to humanizea non-human antibody, as described supra. The framework regions may benaturally occurring or consensus framework regions, and preferably humanframework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479(1998) for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds a polypeptide of theinvention. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substitutions improve binding of the antibody to itsantigen. Additionally, such methods may be used to make amino acidsubstitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855;Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature314:452-454) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-54) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, e.g., a heavy or light chain of anantibody of the invention, requires construction of an expression vectorcontaining a polynucleotide that encodes the antibody. Once apolynucleotide encoding an antibody molecule or a heavy or light chainof an antibody, or portion thereof (preferably containing the heavy orlight chain variable domain), of the invention has been obtained, thevector for the production of the antibody molecule may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing a protein by expressing a polynucleotidecontaining an antibody encoding nucleotide sequence are describedherein. Methods which are well known to those skilled in the art can beused to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination. Theinvention, thus, provides replicable vectors comprising a nucleotidesequence encoding an antibody molecule of the invention, or a heavy orlight chain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., 1986, Gene 45:101; Cockett et al., 1990,Bio/Technology 8:2).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.2:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiationsignals may also be required for efficient translation of insertedantibody coding sequences. These signals include the ATG initiationcodon and adjacent sequences. Furthermore, the initiation codon must bein phase with the reading frame of the desired coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bittner et al., 1987, Methods inEnzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan,1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215); and hygro,which confers resistance to hygromycin (Santerre et al., 1984, Gene30:147). Methods commonly known in the art of recombinant DNA technologywhich can be used are described in Ausubel et al. (eds.), 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, CurrentProtocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin etal., 1981, J. Mol. Biol. 150:1, which are incorporated by referenceherein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol.3:257).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci.USA 77:2197). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

Antibody Conjugates

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20 or 50 amino acids of the polypeptide) of the present invention togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences. The antibodies may bespecific for antigens other than polypeptides (or portion thereof,preferably at least 10, 20 or 50 amino acids of the polypeptide) of thepresent invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are incorporated by reference in their entireties.

The present invention further includes compositions comprising thepolypeptides of the present invention (e.g., those comprising animmunogenic or antigenic epitope) fused or conjugated to heterologouspolypeptide sequences (e.g., antibody domains other than the variableregions). For example, the polypeptides of the present invention may befused or conjugated to an antibody Fc region, or portion thereof. Forexample, polypeptides of the present invention (including fragments orvariants thereof), may be fused with the constant domain ofimmunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2,CH3, or any combination thereof and portions thereof, resulting inchimeric polypeptides. The antibody portion fused to a polypeptide ofthe present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341 (1992) (said references incorporated by reference intheir entireties). By way of another non-limiting example, polypeptidesand/or antibodies of the present invention (including fragments orvariants thereof) may be fused with albumin (including but not limitedto recombinant human serum albumin or fragments or variants thereof(see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, hereinincorporated by reference in their entirety)). In a preferredembodiment, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) are fused with the mature formof human serum albumin (i.e., amino acids 1-585 of human serum albuminas shown in FIGS. 1 and 2 of EP Patent 0 322 094) which is hereinincorporated by reference in its entirety. In another preferredembodiment, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) are fused with polypeptidefragments comprising, or alternatively consisting of, amino acidresidues 1-z of human serum albumin, where z is an integer from 369 to419, as described in U.S. Pat. No. 5,766,883 herein incorporated byreference in its entirety. Polypeptides and/or antibodies of the presentinvention (including fragments or variants thereof) may be fused toeither the N- or C-terminal end of the heterologous protein (e.g.,immunoglobulin Fc polypeptide or human serum albumin polypeptide).Polynucleotides encoding fusion proteins of the invention are alsoencompassed by the invention.

As discussed, supra, the polypeptides of the present invention may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides of the presentinvention may be fused or conjugated to the above antibody portions tofacilitate purification. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (See, e.g., EP 394,827;Traunecker et al., Nature 331:84-86 (1988)). Enhanced delivery of anantigen across the epithelial barrier to the immune system has beendemonstrated for antigens (e.g., insulin) conjugated to an FcRn bindingpartner such as IgG or Fc fragments (see, e.g., PCT Publications WO96/22024 and WO 99/04813). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide-linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995) 0.

Moreover, the polypeptides of the invention (e.g., antibodies orfragments thereof) can be fused to marker sequences, such as a peptideto facilitates their purification. In a further embodiment, nucleicacids encoding the polypeptides of the invention (including, but notlimited to nucleic acids encoding immunogenic and/or antigenic epitopes)can also be recombined with a gene of interest as an epitope tag (e.g.,the hemagglutinin tag (“HA”) or flag tag) to aid in detection andpurification of the expressed polypeptide. In preferred embodiments, themarker amino acid sequence is a hexa-histidine peptide, such as the tagprovided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment, diagnosis, detection,and/or prevention regimen. Detection can be facilitated by coupling theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, radioactive materials,positron emitting metals using various positron emission tomographies,and nonradioactive paramagnetic metal ions. See, for example, U.S. Pat.No. 4,741,900 for metal ions which can be conjugated to antibodies foruse as diagnostics according to the present invention. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ¹¹¹Inor ⁹⁹TC.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion. A cytotoxin orcytotoxic agent includes any agent that is detrimental to cells.Examples include paclitaxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as REM buffer (1% NP-40 or Triton X-100, 1%sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate atpH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest is conjugated to a labeled compound (e.g., 3H or125I) in the presence of increasing amounts of an unlabeled secondantibody.

Therapeutic Uses

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatment,diagnosis, detection, and/or prevention of one or more of the describeddisorders. Therapeutic compounds of the invention include, but are notlimited to, antibodies of the invention (including fragments, analogsand derivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof as described herein). The antibodies of theinvention can be used to treat, diagnose, detect, prevent, and/orinhibit diseases and disorders associated with aberrant expressionand/or activity of a polypeptide of the invention, including, forexample, but not limited to, osteoporosis, disorders in cartilageproduction and/or maintenance, arthritis (e.g., rheumatoid arthritis,and osteoarthritis); regeneration of dentin or bone lost due toperiodontal disease; neurodegenerative diseases; and autoimmune diseasesand/or disorders (e.g., systemic erythromatosus lupus). The treatment,diagnosis, detection, and/or prevention of diseases and disordersassociated with aberrant expression and/or activity of a polypeptide ofthe invention includes, but is not limited to, alleviating symptomsassociated with those diseases and disorders. Antibodies of theinvention may be provided in pharmaceutically acceptable compositions asknown in the art or as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides,including fragments thereof. Preferred binding affinities include thosewith a dissociation constant or Kd less than 5×10-6 M, 10-6 M, 5×10-7 M,10-7 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M, 10-10 M, 5×10-11M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M, 5×10-14 M, 10-14 M,5×10-15 M, and 10-15 M.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,diagnose, detect, prevent, and/or inhibit a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention, by way of gene therapy. Gene therapy refers to therapyperformed by the administration to a subject of an expressed orexpressible nucleic acid. In this embodiment of the invention, thenucleic acids produce their encoded protein that mediates a therapeuticeffect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), 1993, Current Protocols inMolecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY.

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody nucleic acids(Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935;Zijlstra et al., 1989, Nature 342:435-438). In specific embodiments, theexpressed antibody molecule is a single chain antibody; alternatively,the nucleic acid sequences include sequences encoding both the heavy andlight chains, or fragments thereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, DUPONT™), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432)(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180 dated Apr. 16, 1992(Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilson et al.); WO92/20316dated Nov. 26, 1992 (Findeis et al.); WO93/14188 dated Jul. 22, 1993(Clarke et al.), WO 93/20221 dated Oct. 14, 1993 (Young)).Alternatively, the nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., 1993, Meth. Enzymol.217:581-599). These retroviral vectors have been deleted of retroviralsequences that are not necessary for packaging of the viral genome andintegration into host cell DNA. The nucleic acid sequences encoding theantibody to be used in gene therapy are then cloned into one or morevectors, that facilitates delivery of the gene into a patient. Moredetail about retroviral vectors can be found in Boesen et al., 1994,Biotherapy 6:291-302, which describes the use of a retroviral vector todeliver the mdr1 gene to hematopoietic stem cells in order to make thestem cells more resistant to chemotherapy. Other references illustratingthe use of retroviral vectors in gene therapy are: Clowes et al., 1994,J. Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473;Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossmanand Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3:499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5:3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT PublicationWO94/12649; and Wang, et al., 1995, Gene Therapy 2:775-783. In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300;U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644;Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598, dated Apr. 28,1994; Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald, 1980,Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo ClinicProc. 61:771).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Composition

The invention provides methods of treatment, diagnosis, detection,prevention, inhibition, and/or prophylaxis by administration to asubject of an effective amount of a compound or pharmaceuticalcomposition of the invention, preferably an antibody of the invention.In a preferred aspect, the compound is substantially purified (e.g.,substantially free from substances that limit its effect or produceundesired side-effects). The subject is preferably an animal, includingbut not limited to animals such as cows, pigs, horses, chickens, cats,dogs, etc., and is preferably a mammal, and most preferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part ofa retroviral or other vector, etc. Methods of introduction include butare not limited to intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, and oral routes. Thecompounds or compositions may be administered by any convenient route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, it may be desirable to introduce the pharmaceutical compoundsor compositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, 1990, Science249:1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York,pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201;Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J.Med. 321:574). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol.Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al.,1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(1990, Science 249:1527-1533).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, DUPONT™), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, diagnosis, detection, inhibition, and/or prevention of adisease or disorder associated with aberrant expression and/or activityof a polypeptide of the invention can be determined by standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

Antibodies of the present invention may be radiolabeled to be employedin radioimmunotherapy. Antibodies may be used as targeting andpretargeting molecules. Such molecules of the present invention may beradiolabeled by methods well known to those of ordinary skill in theart, which include, but are not limited to, radiolabeled chelation ofthe antibody and antibody phage libraries for targetingradioimmunotherapeutics. See e.g., DeNardo, et al., Clin. Cancer Res.5(10S):3213s-3218s (1999); Quadri, et al., Q. J. Nucl. Med. 42:250-261(1998); the contents of each of which are incorporated by reference inits entirety.

For chelation, different chemical linkages can be inserted between theantibody and the radiolabeled chelate. Radiolabeled monoclonalantibodies reactive with a target antigen can selectively delivercytotoxic or diagnostic isotopes to malignant cells in vivo. Theconstruction of pretargeting molecules can be provided using thediversity and malleability of antibody genes. Diverse arrays of singlechain antibody fragments (i.e., scFvs) can be obtained that are reactivewith a target antigen by selection from human naive phage antibodylibraries. ScFvs can also be cloned directly from hybridoma forconstruction of phage libraries that facilitate subsequent manipulation:e.g., affinity maturation and modification of specificity. ScFvsaffinity selected from these sources to their specific antigen targetshave demonstrated a wide spectrum of binding characteristics. Antibodyheavy (V(H)) and light (V(L)) genes from selected ScFvs may be cloned ascassettes into diabody molecules. This application is discussed further,below, in the method for specific destruction of cells by administeringpolypeptides of the invention in association with toxins or cytotoxicprodrugs.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases and/ordisorders associated with the aberrant expression and/or activity of apolypeptide of the invention. The invention provides for the detectionof aberrant expression of a polypeptide of interest, comprising (a)assaying the expression of the polypeptide of interest in cells or bodyfluid of an individual using one or more antibodies specific to thepolypeptide interest and (b) comparing the level of gene expression witha standard gene expression level, whereby an increase or decrease in theassayed polypeptide gene expression level compared to the standardexpression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, M., et al., J. Cell.Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol.105:3087-3096 (1987)). Other antibody-based methods useful for detectingprotein gene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitableantibody assay labels are known in the art and include enzyme labels,such as, glucose oxidase; radioisotopes, such as iodine (¹³¹I, ¹²⁵I,¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium(^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc),thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶³Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm,¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru;luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

Techniques known in the art may be applied to label antibodies of theinvention. Such techniques include, but are not limited to, the use ofbifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065;5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990;5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contentsof each of which are hereby incorporated by reference in its entirety).

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide of theinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99 mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(SIGMA™, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Immune System-Related Disorders

Diagnosis

The present inventors have discovered that IL17RLP is expressed in adultpulmonary tissue. For a number of immune system-related disorders,substantially altered (increased or decreased) levels of IL17RLP geneexpression can be detected in immune system tissue or other cells orbodily fluids (e.g., sera, plasma, urine, synovial fluid or spinalfluid) taken from an individual having such a disorder, relative to a“standard” IL17RLP gene expression level, that is, the IL17RLPexpression level in immune system tissues or bodily fluids from anindividual not having the immune system disorder. Thus, the inventionprovides a diagnostic method useful during diagnosis of an immune systemdisorder, which involves measuring the expression level of the geneencoding the IL17RLP protein in immune system tissue or other cells orbody fluid from an individual and comparing the measured gene expressionlevel with a standard IL17RLP gene expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of an immune system disorder.

In particular, it is believed that certain tissues in mammals withcancer of the immune system express significantly enhanced levels of theIL17RLP protein and mRNA encoding the IL17RLP protein when compared to acorresponding “standard” level. Further, it is believed that enhancedlevels of the IL17RLP protein can be detected in certain body fluids(e.g., sera, plasma, urine, and spinal fluid) from mammals with such acancer when compared to sera from mammals of the same species not havingthe cancer.

Thus, the invention provides a diagnostic method useful during diagnosisof an immune system disorder, including cancers of this system, whichinvolves measuring the expression level of the gene encoding the IL17RLPprotein in immune system tissue or other cells or body fluid from anindividual and comparing the measured gene expression level with astandard IL17RLP gene expression level, whereby an increase or decreasein the gene expression level compared to the standard is indicative ofan immune system disorder.

Where a diagnosis of a disorder in the immune system including diagnosisof a tumor, has already been made according to conventional methods, thepresent invention is useful as a prognostic indicator, whereby patientsexhibiting enhanced IL17RLP gene expression will experience a worseclinical outcome relative to patients expressing the gene at a levelnearer the standard level.

By “assaying the expression level of the gene encoding the IL17RLPprotein” is intended qualitatively or quantitatively measuring orestimating the level of the IL17RLP protein or the level of the mRNAencoding the IL17RLP protein in a first biological sample eitherdirectly (e.g., by determining or estimating absolute protein level ormRNA level) or relatively (e.g., by comparing to the IL17RLP proteinlevel or mRNA level in a second biological sample). Preferably, theIL17RLP protein level or mRNA level in the first biological sample ismeasured or estimated and compared to a standard IL17RLP protein levelor mRNA level, the standard being taken from a second biological sampleobtained from an individual not having the disorder or being determinedby averaging levels from a population of individuals not having adisorder of the immune system. As will be appreciated in the art, once astandard IL17RLP protein level or mRNA level is known, it can be usedrepeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains IL17RLP protein or mRNA. As indicated, biological samplesinclude body fluids (such as sera, plasma, urine, synovial fluid andspinal fluid) which contain free extracellular domains of IL17RLPprotein, immune system tissue, and other tissue sources found to expresscomplete, mature or extracellular domain of the IL17RLP. Methods forobtaining tissue biopsies and body fluids from mammals are well known inthe art. Where the biological sample is to include mRNA, a tissue biopsyis the preferred source.

The present invention is useful for treatment, diagnosis, detection,and/or prevention of various immune system-related disorders in mammals,preferably humans. Such disorders include tumors, cancers, interstitiallung disease (such as Langerhans cell granulomatosis), and anydisregulation of immune cell function including, but not limited to,autoimmunity, arthritis, leukemias, lymphomas, immunosuppression,immunity, humoral immunity, inflammatory bowel disease, myelosuppression, and the like.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described byChomczynski and Sacchi (Anal. Biochem. 162:156-159 (1987)). Levels ofmRNA encoding the IL17RLP protein are then assayed using any appropriatemethod. These include Northern blot analysis, S1 nuclease mapping, thepolymerase chain reaction (PCR), reverse transcription in combinationwith the polymerase chain reaction (RT-PCR), and reverse transcriptionin combination with the ligase chain reaction (RT-LCR).

Assaying IL17RLP protein levels in a biological sample can occur usingantibody-based techniques. For example, IL17RLP protein expression intissues can be studied with classical immunohistological methods(Jalkanen, M., et al., J. Cell Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-basedmethods useful for detecting IL17RLP protein gene expression includeimmunoassays, such as the enzyme linked immunosorbent assay (ELISA) andthe radioimmunoassay (RIA). Suitable antibody assay labels are known inthe art and include enzyme labels, such as, glucose oxidase, andradioisotopes, such as iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In),and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga),palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F),¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru; luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin.

Techniques known in the art may be applied to label antibodies of theinvention. Such techniques include, but are not limited to, the use ofbifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065;5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990;5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contentsof each of which are hereby incorporated by reference in its entirety).

In addition to assaying IL17RLP protein levels in a biological sampleobtained from an individual, IL17RLP protein can also be detected invivo by imaging. Antibody labels or markers for in vivo imaging ofIL17RLP protein include those detectable by X-radiography, NMR or ESR.For X-radiography, suitable labels include radioisotopes such as bariumor cesium, which emit detectable radiation but are not overtly harmfulto the subject Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma.

Antibody labels or markers for in vivo imaging of IL17RLP polypeptideinclude those detectable by X-radiography, NMR, MRI, CAT-scans or ESR.For X-radiography, suitable labels include radioisotopes such as bariumor cesium, which emit detectable radiation but are not overtly harmfulto the subject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma. Where in vivo imaging is used to detect enhanced levels ofIL17RLP polypeptide for diagnosis in humans, it may be preferable to usehuman antibodies or “humanized” chimeric monoclonal antibodies. Suchantibodies can be produced using techniques described herein orotherwise known in the art. For example methods for producing chimericantibodies are known in the art. See, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne at al., Nature 312:643 (1984); Neuberger et al., Nature314:268 (1985).

Additionally, any IL17RLP polypeptide whose presence can be detected,can be administered. For example, IL17RLP polypeptides labeled with aradio-opaque or other appropriate compound can be administered andvisualized in vivo, as discussed, above for labeled antibodies. Furthersuch IL17RLP polypeptides can be utilized for in vitro diagnosticprocedures.

An IL17RLP polypeptide-specific antibody or antibody fragment which hasbeen labeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc, (¹³¹I, ¹²⁵I, ¹²³I,¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^(115m)In,^(113m)In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium(²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo),xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb,¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for immune systemdisorder. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ^(99m)Tc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain IL17RLP protein. Invivo tumor imaging is described by Burchiel and coworkers (Chapter 13 inTumor Imaging: The Radiochemical Detection of Cancer, Burchiel, S. W.and Rhodes, B. A., eds., Masson Publishing Inc. (1982)).

Treatment

As noted above, IL17RLP polynucleotides and polypeptides are useful fordiagnosis of conditions involving abnormally high or low expression ofIL17RLP activities. Given the cells and tissues where IL17RLP isexpressed as well as the activities modulated by IL17RLP, it is readilyapparent that a substantially altered (increased or decreased) level ofexpression of IL17RLP in an individual compared to the standard or“normal” level produces pathological conditions related to the bodilysystem(s) in which IL17RLP is expressed and/or is active.

It will also be appreciated by one of ordinary skill that, since theIL17RLP protein of the invention is a member of the interleukin (IL)-17receptor family, the extracellular domain of the protein may be releasedin soluble form from the cells which express the IL17RLP by proteolyticcleavage. Therefore, when IL17RLP soluble extracellular domain is addedfrom an exogenous source to cells, tissues or the body of an individual,the protein will exert its physiological activities on its target cellsof that individual. Also, cells expressing this transmembrane proteinmay be added to cells, tissues or the body of an individual and theseadded cells will bind to cells expressing IL17RLP, whereby the cellsexpressing IL17RLP can cause actions (e.g. cell stimulation) on theligand-bearing target cells.

Therefore, it will be appreciated that conditions caused by a decreasein the standard or normal level of IL17RLP activity in an individual,particularly disorders of the immune system, can be treated, diagnosed,detected, and/or prevented by administration of IL17RLP polypeptide (inthe form of a soluble extracellular domain or cells expressing thecomplete protein). Thus, the invention also provides a method oftreatment, diagnosis, detection, and/or prevention of an individual inneed of an increased level of IL17RLP activity comprising administeringto such an individual a pharmaceutical composition comprising an amountof an isolated IL17RLP polypeptide of the invention, particularly anextracellular domain of the IL17RLP protein of the invention, effectiveto increase the IL17RLP activity level in such an individual.

Since IL17RLP is a novel homologue of the recently described IL-17receptor, it will have a wide range of cytokine receptor-likeactivities. IL17RLP, or agonists of IL17RLP, may be employed to enhancehost defenses against resistant chronic and acute infections, forexample, mycobacterial infections via the attraction and activation ofmicrobicidal leukocytes. IL17RLP may also be employed to increase T-cellproliferation by the stimulation of IL-2 biosynthesis for the treatment,diagnosis, detection, and/or prevention of T-cell mediated auto-immunediseases and lymphocytic leukemias. IL17RLP may also be employed toregulate hematopoiesis, by regulating the activation and differentiationof various hematopoietic progenitor cells, for example, to releasemature leukocytes from the bone marrow following chemotherapy, i.e., instem cell mobilization. IL17RLP may also be employed to treat, diagnose,detect, and/or prevent sepsis. Soluble IL17RLP extracellular domains maybe used as antagonists for IL17RLP activity, and, as such, will beuseful therapeutically, as a mechanism to regulate the activity ofendogenous IL17RLP. Also, stimulation of IL17RLP strongly induces IL-6expression. IL-6 is a potent growth factor for myelomas, plasmacytomas,and hybridomas and is involved in the growth of Lennert's LymphomaT-cells. As a result, IL17RLP agonists and soluble IL17RLP extracellulardomains may be used in the treatment, diagnosis, detection, and/orprevention of such cancers, analogous disease states, and others knownto those of skill in the art.

IL17RLP polynucleotides or polypeptides, or agonists of IL17RLP, can beused in the treatment, diagnosis, detection, and/or prevention ofinfectious agents. For example, by increasing the immune response,particularly increasing the proliferation and differentiation of T or Bcells, infectious diseases may be treated, diagnosed, detected, and/orprevented. The immune response may be increased by either enhancing anexisting immune response, or by initiating a new immune response.Alternatively, IL17RLP polynucleotides or polypeptides, or agonists orantagonists of IL17RLP, may also directly inhibit the infectious agent,without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated, diagnosed, detected, and/or prevented byIL17RLP polynucleotides or polypeptides, or agonists of IL17RLP.Examples of viruses, include, but are not limited to the following DNAand RNA viruses and viral families: Arbovirus, Adenoviridae,Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae,Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae,Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus,Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae,Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A,Influenza B, and parainfluenza), Papiloma virus, Papovaviridae,Parvoviridae, Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia),Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling withinthese families can cause a variety of diseases or symptoms, including,but not limited to: arthritis, bronchiollitis, respiratory syncytialvirus, encephalitis, eye infections (e.g., conjunctivitis, keratitis),chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta),Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellowfever, meningitis, opportunistic infections (e.g., AIDS), pneumonia,Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia. IL17RLP polynucleotides or polypeptides, or agonists orantagonists of IL17RLP, can be used to treat, prevent, diagnose, and/ordetect any of these symptoms or diseases. In specific embodiments,IL17RLP polynucleotides, polypeptides, or agonists are used to treat,prevent, detect, and/or diagnose: meningitis, Dengue, EBV, and/orhepatitis (e.g., hepatitis B). In an additional specific embodimentIL17RLP polynucleotides, polypeptides, or agonists are used to treatpatients nonresponsive to one or more other commercially availablehepatitis vaccines. In a further specific embodiment, IL17RLPpolynucleotides, polypeptides, or agonists are used to treat, prevent,detect, and/or diagnose AIDS. In an additional specific embodimentIL17RLP polynucleotides, polypeptides, agonists, and/or antagonists areused to treat, prevent, detect, and/or diagnose patients withcryptosporidiosis.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated, diagnosed, detected, and/or prevented byIL17RLP polynucleotides or polypeptides, or agonists or antagonists ofIL17RLP, include, but not limited to, the following Gram-Negative andGram-positive bacteria and bacterial families and fungi: Actinomycetales(e.g., Corynebacterium, Mycobacterium, Norcardia), Cryptococcusneoformans, Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium),Bacteroidaceae, Blastomycosis, Bordetella, Borrelia (e.g., Borreliaburgdorferi, Brucellosis, Candidiasis, Campylobacter,Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E. coli (e.g.,Enterotoxigenic E. coli and Enterohemorrhagic E. coli),Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, andSalmonella paratyphi), Serratia, Yersinia), Erysipelothrix,Helicobacter, Legionellosis, Leptospirosis, Listeria (e.g., Listeriamonocytogenes), Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae,Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Meisseriameningitidis, Pasteurellacea Infections (e.g., Actinobacillus,Heamophilus (e.g., Heamophilus influenza type B), Pasteurella),Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp.,Staphylococcal, Meningiococcal, Pneumococcal and Streptococcal (e.g.,Streptococcus pneumoniae and Group B Streptococcus). These bacterial orfungal families can cause the following diseases or symptoms, including,but not limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. IL17RLP polynucleotides or polypeptides, or agonistsor antagonists of IL17RLP, can be used to treat, prevent, diagnose,and/or detect any of these symptoms or diseases. In specificembodiments, IL17RLP polynucleotides, polypeptides, or agonists thereofare used to treat, prevent, detect, and/or diagnose: tetanus, Diptheria,botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can betreated, diagnosed, detected, and/or prevented by IL17RLPpolynucleotides or polypeptides, or agonists of IL17RLP, include, butnot limited to, the following families or class: Amebiasis, Babesiosis,Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic,Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax,Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). Theseparasites can cause a variety of diseases or symptoms, including, butnot limited to: Scabies, Trombiculiasis, eye infections, intestinaldisease (e.g., dysentery, giardiasis), liver disease, lung disease,opportunistic infections (e.g., AIDS related), malaria, pregnancycomplications, and toxoplasmosis. IL17RLP polynucleotides orpolypeptides, or agonists or antagonists of IL17RLP, can be used totreat, prevent, diagnose, and/or detect any of these symptoms ordiseases. In specific embodiments, IL17RLP polynucleotides,polypeptides, or agonists thereof are used to treat, prevent, detect,and/or diagnose malaria.

In another embodiment, the invention provides a method of deliveringcompositions containing the polypeptides of the invention (e.g.,compositions containing IL17RLP polypeptides or anti-IL17RLP antibodiesassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs) to targeted cells, such as, for example, B or Tcells expressing IL17RLP. IL17RLP polypeptides or anti-IL17RLPantibodies of the invention may be associated with heterologouspolypeptides, heterologous nucleic acids, toxins, or prodrugs viahydrophobic, hydrophilic, ionic and/or covalent interactions.

In one embodiment, the invention provides a method for the specificdelivery of compositions of the invention to cells by administeringpolypeptides of the invention (e.g., IL17RLP polypeptides oranti-IL17RLP antibodies) that are associated with heterologouspolypeptides or nucleic acids. In one example, the invention provides amethod for delivering a therapeutic protein into the targeted cell. Inanother example, the invention provides a method for delivering a singlestranded nucleic acid (e.g., antisense or ribozymes) or double strandednucleic acid (e.g., DNA that can integrate into the cell's genome orreplicate episomally and that can be transcribed) into the targetedcell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., IL17RLP polypeptidesor anti-IL17RLP antibodies) in association with toxins or cytotoxicprodrugs.

In a specific embodiment, the invention provides a method for thespecific destruction of cells of T or B cell lineage (e.g., T or B cellrelated leukemias or lymphomas) by administering IL17RLP polypeptides inassociation with toxins or cytotoxic prodrugs.

In another specific embodiment, the invention provides a method for thespecific destruction of cells of monocytic lineage (e.g., monocyticleukemias or lymphomas) by administering anti-IL17RLP antibodies inassociation with toxins or cytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. “Toxin” also includes a cytostatic orcytocidal agent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, ²¹³Bi, or other radioisotopes suchas, for example, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S,⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁵Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, ⁹⁰Yttrium, ¹¹⁷Tin,¹⁸⁶Rhenium, ¹⁶⁶Holmium, and ¹⁸⁸Rhenium; luminescent labels, such asluminol; and fluorescent labels, such as fluorescein and rhodamine, andbiotin.

Techniques known in the art may be applied to label antibodies of theinvention. Such techniques include, but are not limited to, the use ofbifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065;5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990;5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contentsof each of which are hereby incorporated by reference in its entirety).A cytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

By “cytotoxic prodrug” is meant a non-toxic compound that is convertedby an enzyme, normally present in the cell, into a cytotoxic compound.Cytotoxic prodrugs that may be used according to the methods of theinvention include, but are not limited to, glutamyl derivatives ofbenzoic acid mustard alkylating agent, phosphate derivatives ofetoposide or mitomycin C, cytosine arabinoside, daunorubisin, andphenoxyacetamide derivatives of doxorubicin.

An additional condition, disease or symptom that can be treated,prevented, detected, and/or diagnosed by IL17RLP polynucleotides orpolypeptides, or agonists of IL17RLP, is osteomyelitis.

An additional condition, disease or symptom that can be treated,prevented, detected, and/or diagnosed by IL17RLP polynucleotides orpolypeptides, or agonists of IL17RLP, is endocarditis.

Preferably, treatment, diagnosis, detection, and/or prevention usingIL17RLP polynucleotides or polypeptides, or agonists of IL17RLP, couldeither be by administering an effective amount of IL17RLP polypeptide tothe patient, or by removing cells from the patient, supplying the cellswith IL17RLP polynucleotide, and returning the engineered cells to thepatient (ex vivo therapy). Moreover, as further discussed herein, theIL17RLP polypeptide or polynucleotide can be used as an adjuvant in avaccine to raise an immune response against infectious disease.

In a specific embodiment, IL17RLP polynucleotides or polypeptides, oragonists thereof (e.g., anti-IL17RLP antibodies) are used to treat,diagnose, detect, and/or prevent a disorder characterized by deficientserum immunoglobulin production, recurrent infections, and/or immunesystem dysfunction. Moreover, IL17RLP polynucleotides or polypeptides,or agonists thereof (e.g., anti-IL17RLP antibodies) may be used totreat, diagnose, detect, and/or prevent infections of the joints, bones,skin, and/or parotid glands, blood-borne infections (e.g., sepsis,meningitis, septic arthritis, and/or osteomyelitis), autoimmune diseases(e.g., those disclosed herein), inflammatory disorders, andmalignancies, and/or any disease or disorder or condition associatedwith these infections, diseases, disorders and/or malignancies)including, but not limited to, CVID, other primary immune deficiencies,HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media,conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g.,severe herpes zoster), and/or pheumocystis carnii.

Additional preferred embodiments of the invention include, but are notlimited to, the use of IL17RLP polypeptides, IL17RLP polynucleotides,and functional agonists thereof, in the applications that follow below.

As a chemoattractant of neutrophils. In a preferred embodiment, IL17RLPpolypeptides, polynucleotides, agonists, and/or antagonists thereof maybe used as a chemoattractant of neutrophils in the spinal cord.

As a means of stimulating bone and/or cartilage cell growth. Thus,IL17RLP polypeptides, polynucleotides, agonists, and/or antagoniststhereof may be useful, for example, in osteoporosis, cartilageproduction, osteoarthritis, and/or regeneration of dentin and/or bonelost due to periodontal disease.

As a means of induction of TNF-alpha, IL-1beta, and/or IL-6 expression.

As an agent of regulation and/or maintenance of the growth state and/orcellular activity of cells of the spinal cord. In preferred embodiments,as an agent of regulation and/or maintenance of the growth state and/orcellular activity of glial cells and/or neurons. Thus, IL17RLPpolypeptides, polynucleotides, agonists, and/or antagonists thereof maybe useful, for example, to treat, diagnose, prevent, and/or detectneurodegenerative diseases including amyotrophic lateral sclerosis(ALS); demyelinating diseases including multiple sclerosis; peripheralneuropathies (e.g., Charcot-Marie Tooth (CMT) disease); sensoryneuropathis; neuroallergy; neuroarthropathy; neuroblastoma; olfactoryneuroblastoma; neurochorioretinitis; neurochoroiditis; neurocirculatoryasthenia (e.g., DaCosta's syndrome; effort syndrome; irritable heart;soldier's heart); neurocristopathy; neurocutaneous melanosis;neurocutaneous syndrome; neurocytolysis; neurocytoma; ganglioneuroma;cataracta neurodermatica; atopic dermatitis; neurodermatitis (e.g.,atopic dermatitis; chronic lichen simplex); neurodynia; neuralgia;neuroencephalomyelopathy; neurofibrillary degeneration; Alzheimer'sdisease; neurofibroma; fibroneuroma; schwannoma; plexiform neurofibroma;storiform neurofibroma; neurofibromatosis (e.g., von Recklinghausen'sdisease); abortive neurofibromatosis; neurogenic atrophy; neurogenicbladder, neuroglia (e.g., Kolliker's reticulum); neurogliomatosis;neuroleptanalgesia; neuroleptic malignant syndrome; neurolymphomatosis(e.g., neurolymphomatosis gallinarum); neurolymphomatosis gallinarum;neurolysis; rolling disease; neuroma (e.g., acoustic neuroma; amputationneuroma; neuroma cutis; false neuroma; fibrillary neuroma; plexiformneuroma; neuroma telangiectodes; traumatic neuroma; Vemeuil's neuroma);neuroma cutis; fibrillary neuroma (e.g., plexiform neurofibroma);neuromalacia; elephantiasis neuromatosa; neuromatosis (e.g., as inneurofibromatosis); neuromimesis; neuromyasthenia (e.g., epidemicneuromyasthenia); neuromyelitis (e.g., myeloneuritis; neuromyelitisoptica); neuromyopathy (e.g., carcinomatous neuromyopathy);neuromyositis; neuronitis; neuronopathy (e.g., sensory neuronopathy);neuropapillitis; neuroparalysis (e.g., neuroparalytic keratitis;neuroparalytic ophthalmia); neuropathic albuminuria; neuropathicarthritis (e.g., Charcot's joint; neuropathic arthritis; neuropathicarthropathy; tabetic arthropathy); papilloma neuropathicum; neuropathy(e.g., asymmetric motor neuropathy; brachial plexus neuropathy; diabeticneuropathy; diphtheritic neuropathy; entrapment neuropathy; familialamyloid neuropathy; giant axonal neuropathy; hereditary hypertrophicneuropathy; hereditary sensory radicular, neuropathy; hypertrophicinterstitial neuropathy; ischemic optic neuropathy; isoniazidneuropathy; lead neuropathy; leprous neuropathy; motor dapsoneneuropathy; onion bulb neuropathy; segmental neuropathy; symmetricdistal neuropathy; vitamin B12 neuropathy); neurophonia; neuroplegic;neuropsychologic disorder, neuropsychopathy; neurorelapse;neuroretinitis; neurosarcoidosis; neuroschwannoma (e.g., neurilemoma);neurosis; neurospasm; neurosthenia; neurosyphilis; neurotabes (e.g.,Dejerine's peripheral neurotabes); neurothekeoma; alopecia neurotica;lipomatosis neurotics; neurotrauma; neurotrophic atrophy (e.g., neuriticatrophy); neurotrosis; neurovaricosis; and/or other diseases/disordersof the spinal cord.

IL17RLP polypeptides, polynucleotides, agonists, and/or antagoniststhereof may be useful, for example, to treat, diagnose, prevent, and/ordetect immune and/or autoimmune diseases and disorders and/or conditionsassociated therewith. Thus, IL17RLP polypeptides, polynucleotides,agonists, and/or antagonists thereof may be useful, for example, totreat, diagnose, prevent, and/or detect immune complex disease; immunecomplex disorder; immune complex nephritis; immunodeficiency; immunehemolysis (e.g., conditioned hemolysis); immune inflammation; immunethrombocytopenia; immune thrombocytopenic purpura; immunoblasticlymphadenopathy; immunoblastic lymphoma; immunoblastic sarcoma;immunodeficiency (e.g., cellular immunodeficiency with abnormalimmunoglobulin synthesis; combined immunodeficiency; common variableimmunodeficiency; immunodeficiency with; hypoparathyroidism; phagocyticdysfunction disorders; immunodeficiency; secondary immunodeficiency;severe combined immunodeficiency); immunodeficiency withhypoparathyroidism; immunodeficiency syndrome; immunoproliferativedisorders; immunoproliferative small intestinal disease (e.g.,Mediterranean lymphoma); chronic discoid lupus erythematosus; discoidlupus erythematosus; disseminated lupus erythematosus; lupuserythematodes; lupus erythematosus; lupus erythematosus profundus; lupushypertrophicus; lupus livido; lupus lymphaticus; lupus miliarisdisseminatus faciei; lupus mutilans; lupus papillomatosus; lupus pernio;lupus psoriasis; lupus sclerosus; lupus sebaceus; lupus serpiginosus;lupus superficialis; systemic lupus erythematosus; lupus tuberculosus;lupus tumidus; lupus verrucosus; lupus vulgaris; lupus vulgariserythematoides; acute rheumatic arthritis; atrophic arthritis;chlamydial arthritis; chronic absorptive arthritis; chylous arthritis;arthritis deformans; degenerative arthritis; enteropathic arthritis;filarial arthritis; gouty arthritis; hemophilic arthritis; hypertrophicarthritis; Jaccoud's arthritis; juvenile arthritis; Lyme arthritis;arthritis mutilans; neonatal arthritis of foals; neuropathic arthritis;arthritis nodosa; ochronotic arthritis; proliferative arthritis;psoriatic arthritis; rheumatoid arthritis; suppurative arthritis; and/orarthritis uratica.

Administration to an animal (e.g., mouse, rat, rabbit, hamster, guineapig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat,non-human primate, and human, most preferably human) to boost the immunesystem to produce increased quantities of one or more antibodies (e.g.,IgG, IgA, IgM, and IgE), to induce higher affinity antibody production(e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.

Administration to an animal (including, but not limited to, those listedabove, and also including transgenic animals) incapable of producingfunctional endogenous antibody molecules or having an otherwisecompromised endogenous immune system, but which is capable of producinghuman immunoglobulin molecules by means of a reconstituted or partiallyreconstituted immune system from another animal (see, e.g., publishedPCT Application Nos. WO98/24893, WO/9634096, WO/9633735, and WO/9110741.

A vaccine adjuvant that enhances immune responsiveness to specificantigen. In a specific embodiment, the vaccine adjuvant is an IL17RLPpolypeptide described herein. In another specific embodiment, thevaccine adjuvant is an IL17RLP polynucleotide described herein (i.e.,the IL17RLP polynucleotide is a genetic vaccine adjuvant). As discussedherein, IL17RLP polynucleotides may be administered using techniquesknown in the art, including but not limited to, liposomal delivery,recombinant vector delivery, injection of naked DNA, and gene gundelivery.

An adjuvant to enhance tumor-specific immune responses.

An adjuvant to enhance anti-viral immune responses. Anti-viral immuneresponses that may be enhanced using the compositions of the inventionas an adjuvant, include virus and virus associated diseases or symptomsdescribed herein or otherwise known in the art. In specific embodiments,the compositions of the invention are used as an adjuvant to enhance animmune response to a virus, disease, or symptom selected from the groupconsisting of AIDS, meningitis, Dengue, EBV, and hepatitis (e.g.,hepatitis B). In another specific embodiment, the compositions of theinvention are used as an adjuvant to enhance an immune response to avirus, disease, or symptom selected from the group consisting ofHIV/AIDS, Respiratory syncytial virus, Dengue, Rotavirus, Japanese Bencephalitis, Influenza A and B, Parainfluenza, Measles,Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever, Herpessimplex, and yellow fever. In another specific embodiment, thecompositions of the invention are used as an adjuvant to enhance animmune response to the HIV gp120 antigen.

An adjuvant to enhance anti-bacterial or anti-fungal immune responses.Anti-bacterial or anti-fungal immune responses that may be enhancedusing the compositions of the invention as an adjuvant, include bacteriaor fungus and bacteria or fungus associated diseases or symptomsdescribed herein or otherwise known in the art. In specific embodiments,the compositions of the invention are used as an adjuvant to enhance animmune response to a bacteria or fungus, disease, or symptom selectedfrom the group consisting of tetanus, Diphtheria, botulism, andmeningitis type B. In another specific embodiment, the compositions ofthe invention are used as an adjuvant to enhance an immune response to abacteria or fungus, disease, or symptom selected from the groupconsisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi,Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae,Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli,Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium(malaria).

An adjuvant to enhance anti-parasitic immune responses. Anti-parasiticimmune responses that may be enhanced using the compositions of theinvention as an adjuvant, include parasite and parasite associateddiseases or symptoms described herein or otherwise known in the art. Inspecific embodiments, the compositions of the invention are used as anadjuvant to enhance an immune response to a parasite. In anotherspecific embodiment, the compositions of the invention are used as anadjuvant to enhance an immune response to Plasmodium (malaria).

As a stimulator of B cell responsiveness to pathogens.

As an agent that elevates the immune status of an individual prior totheir receipt of immunosuppressive therapies.

As an agent to induce higher affinity antibodies.

As an agent to increase serum immunoglobulin concentrations.

As an agent to accelerate recovery of immunocompromised individuals.

As an agent to boost immunoresponsiveness among aged populations.

As an immune system enhancer prior to, during, or after bone marrowtransplant and/or other transplants (e.g., allogeneic or xenogeneicorgan transplantation). With respect to transplantation, compositions ofthe invention may be administered prior to, concomitant with, and/orafter transplantation. In a specific embodiment, compositions of theinvention are administered after transplantation, prior to the beginningof recovery of T-cell populations. In another specific embodiment,compositions of the invention are first administered aftertransplantation after the beginning of recovery of T cell populations,but prior to full recovery of B cell populations.

As an agent to boost immunoresponsiveness among B cell immunodeficientindividuals, such as, for example, an individual who has undergone apartial or complete splenectomy. B cell immunodeficiencies that may beameliorated, treated, diagnosed, detected, and/or prevented byadministering the IL17RLP polypeptides or polynucleotides of theinvention, or agonists thereof, include, but are not limited to, severecombined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosinedeaminase deficiency (ADA deficiency), X-linked agammaglobulinemia(XLA), Bruton's disease, congenital agammaglobulinemia, X-linkedinfantile agammaglobulinemia, acquired agammaglobulinemia, adult onsetagammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia,hypogammaglobulinemia, transient hypogammaglobulinemia of infancy,unspecified hypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CV1) (acquired), Wiskott-Aldrich Syndrome (WAS),X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiencywith hyper IgM, selective IgA deficiency, IgG subclass deficiency (withor without IgA deficiency), antibody deficiency with normal or elevatedIgs, immunodeficiency with thymoma, Ig heavy chain deletions, kappachain deficiency, B cell lymphoproliferative disorder (BLPD), selectiveIgM immunodeficiency, recessive agammaglobulinemia (Swiss type),reticular dysgenesis, neonatal neutropenia, severe congenitalleukopenia, thymic alymophoplasia-aplasia or dysplasia withimmunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linkedlymphoproliferative syndrome (XLP), Nezelof syndrome-combinedimmunodeficiency with Igs, purine nucleoside phosphorylase deficiency(PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severecombined immunodeficiency.

As an agent to boost immunoresponsiveness among individuals having anacquired loss of B cell function. Conditions resulting in an acquiredloss of B cell function that may be ameliorated, treated, diagnosed,detected, and/or prevented by administering the IL17RLP polypeptides orpolynucleotides of the invention, or agonists thereof, include, but arenot limited to, HIV Infection, AIDS, bone marrow transplant, and B cellchronic lymphocytic leukemia (CLL).

As an agent to boost immunoresponsiveness among individuals having atemporary immune deficiency. Conditions resulting in a temporary immunedeficiency that may be ameliorated, treated, diagnosed, detected, and/orprevented by administering the IL17RLP polypeptides or polynucleotidesof the invention, or agonists thereof, include, but are not limited to,recovery from viral infections (e.g., influenza), conditions associatedwith malnutrition, recovery from infectious mononucleosis, or conditionsassociated with stress, recovery from measles, recovery from bloodtransfusion, recovery from surgery.

As a regulator of antigen presentation by monocytes, dendritic cells,and/or B-cells. In one embodiment, IL17RLP polypeptides (in soluble,membrane-bound or transmembrane forms) or polynucleotides enhanceantigen presentation or antagonize antigen presentation in vitro or invivo. Moreover, in related embodiments, said enhancement orantagonization of antigen presentation may be useful as an anti-tumortreatment or to modulate the immune system.

As an agent to direct an individual's immune system towards developmentof a humoral response (i.e. TH2) as opposed to a TH1 cellular response.

As a means to induce tumor proliferation and thus make it moresusceptible to anti-neoplastic agents. For example, multiple myeloma isa slowly dividing disease and is thus refractory to virtually allanti-neoplastic regimens. If these cells were forced to proliferate morerapidly their susceptibility profile would likely change.

As a therapy for generation and/or regeneration of lymphoid tissuesfollowing surgery, trauma or genetic defect.

As a gene-based therapy for genetically inherited disorders resulting inimmuno-incompetence such as observed among SCID patients.

As an antigen for the generation of antibodies to inhibit or enhanceIL17RLP mediated responses.

As a means of activating monocytes/macrophages to defend againstparasitic diseases that effect monocytes such as Leshmania.

As pretreatment of bone marrow samples prior to transplant. Suchtreatment would increase B cell representation and thus acceleraterecover.

As a means of regulating secreted cytokines that are elicited byIL17RLP.

IL17RLP polypeptides or polynucleotides of the invention, or agonistsmay be used to modulate IgE concentrations in vitro or in vivo.

Additionally, IL17RLP polypeptides or polynucleotides of the invention,or agonists thereof, may be used to treat, prevent, detect, and/ordiagnose IgE-mediated allergic reactions. Such allergic reactionsinclude, but are not limited to, asthma, rhinitis, and eczema.

In a specific embodiment, IL17RLP polypeptides or polynucleotides of theinvention, or agonists thereof, is administered to treat, prevent,diagnose, and/or ameliorate selective IgA deficiency.

In another specific embodiment, IL17RLP polypeptides or polynucleotidesof the invention, or agonists thereof, is administered to treat,prevent, diagnose, and/or ameliorate ataxia-telangiectasia.

In another specific embodiment, IL17RLP polypeptides or polynucleotidesof the invention, or agonists thereof, is administered to treat,prevent, diagnose, and/or ameliorate common variable immunodeficiency.

In another specific embodiment, IL17RLP polypeptides or polynucleotidesof the invention, or agonists thereof, is administered to treat,prevent, diagnose, and/or ameliorate X-linked agammaglobulinemia.

In another specific embodiment, IL17RLP polypeptides or polynucleotidesof the invention, or agonists thereof, is administered to treat,prevent, diagnose, and/or ameliorate severe combined immunodeficiency(SCID).

In another specific embodiment, IL17RLP polypeptides or polynucleotidesof the invention, or agonists thereof, is administered to treat,prevent, diagnose, and/or ameliorate Wiskott-Aldrich syndrome.

In another specific embodiment, IL17RLP polypeptides or polynucleotidesof the invention, or agonists thereof, is administered to treat,prevent, diagnose, and/or ameliorate X-linked Ig deficiency with hyperIgM.

In another specific embodiment, IL17RLP polypeptides or polynucleotidesof the invention, or agonists or antagonists (e.g., anti-IL17RLPantibodies) thereof, is administered to treat, prevent, detect, and/ordiagnose chronic myelogenous leukemia, acute myelogenous leukemia,leukemia, hystiocytic leukemia, monocytic leukemia (e.g., acutemonocytic leukemia), leukemic reticulosis, Shilling Type monocyticleukemia, and/or other leukemias derived from monocytes and/or monocyticcells and/or tissues.

In another specific embodiment, IL17RLP polypeptides or polynucleotidesof the invention, or agonists thereof, is administered to treat,prevent, diagnose, and/or ameliorate monocytic leukemoid reaction, asseen, for example, with tuberculosis.

In another specific embodiment, IL17RLP polypeptides or polynucleotidesof the invention, or agonists thereof, is administered to treat,prevent, diagnose, and/or ameliorate monocytic leukocytosis, monocyticleukopenia, monocytopenia, and/or monocytosis.

In a specific embodiment, IL17RLP polynucleotides or polypeptides of theinvention, and/or anti-IL17RLP antibodies and/or agonists or antagoniststhereof, are used to treat, prevent, detect, and/or diagnose primary Blymphocyte disorders and/or diseases, and/or conditions associatedtherewith. In one embodiment, such primary B lymphocyte disorders,diseases, and/or conditions are characterized by a complete or partialloss of humoral immunity. Primary B lymphocyte disorders, diseases,and/or conditions associated therewith that are characterized by acomplete or partial loss of humoral immunity and that may be prevented,treated, detected and/or diagnosed with compositions of the inventioninclude, but are not limited to, X-Linked Agammaglobulinemia (XLA),severe combined immunodeficiency disease (SCID), and selective IgAdeficiency.

In a preferred embodiment, IL17RLP polynucleotides, polypeptides, and/oragonists and/or antagonists thereof are used to treat, prevent, and/ordiagnose diseases or disorders affecting or conditions associated withany one or more of the various mucous membranes of the body. Suchdiseases or disorders include, but are not limited to, for example,mucositis, mucoclasis, mucocolitis, mucocutaneous leishmaniasis (suchas, for example, American leishmaniasis, leishmaniasis americana,nasopharyngeal leishmaniasis, and New World leishmaniasis),mucocutaneous lymph node syndrome (for example, Kawasaki disease),mucoenteritis, mucoepidermoid carcinoma, mucoepidermoid tumor,mucoepithelial dysplasia, mucoid adenocarcinoma, mucoid degeneration,myxoid degeneration; myxomatous degeneration; myxomatosis, mucoid medialdegeneration (for example, cystic medial necrosis), mucolipidosis(including, for example, mucolipidosis I, mucolipidosis H, mucolipidosisIII, and mucolipidosis IV), mucolysis disorders, mucomembranousenteritis, mucoenteritis, mucopolysaccharidosis (such as, for example,type I mucopolysaccharidosis (i.e., Hurler's syndrome), type ISmucopolysaccharidosis (i.e., Scheie's syndrome or type Vmucopolysaccharidosis), type II mucopolysaccharidosis (i.e., Hunter'ssyndrome), type III mucopolysaccharidosis (i.e., Sanfilippo's syndrome),type IV mucopolysaccharidosis (i.e., Morquio's syndrome), type VImucopolysaccharidosis (i.e., Maroteaux-Lamy syndrome), type VIImucopolysaccharidosis (i.e, mucopolysaccharidosis due tobeta-glucuronidase deficiency), and mucosulfatidosis),mucopolysacchariduria, mucopurulent conjunctivitis, mucopus,mucormycosis (i.e., zygomycosis), mucosal disease (i.e., bovine virusdiarrhea), mucous colitis (such as, for example, mucocolitis andmyxomembranous colitis), and mucoviscidosis (such as, for example,cystic fibrosis, cystic fibrosis of the pancreas, Clarke-Hadfieldsyndrome, fibrocystic disease of the pancreas, mucoviscidosis, andviscidosis). In a highly preferred embodiment, IL17RLP polynucleotides,polypeptides, and/or agonists and/or antagonists thereof are used totreat, prevent, and/or diagnose mucositis, especially as associated withchemotherapy.

In a preferred embodiment, IL17RLP polynucleotides, polypeptides, and/oragonists and/or antagonists thereof are used to treat, prevent, and/ordiagnose diseases or disorders affecting or conditions associated withsinusitis.

All of the above described applications as they may apply to veterinarymedicine.

Antagonists of IL17RLP include binding and/or inhibitory antibodies,antisense nucleic acids, ribozymes or soluble forms (e.g., a solubleextracellular domain) of IL17RLP. These would be expected to reversemany of the activities of the ligand described above as well as findclinical or practical application as:

A means of blocking various aspects of immune responses to foreignagents or self. Examples include autoimmune disorders such as lupus, andarthritis, as well as immunoresponsiveness to skin allergies,inflammation, bowel disease, injury and pathogens.

A therapy for preventing the T or B cell proliferation and Ig secretionassociated with autoimmune diseases such as idiopathic thrombocytopenicpurpura, systemic lupus erythramatosus and MS.

An inhibitor of graft versus host disease or transplant rejection.

A therapy for B cell malignancies such as ALL, Hodgkins disease,non-Hodgkins lymphoma, Chronic lymphocyte leukemia, plasmacytomas,multiple myeloma, Burkitt's lymphoma, and EBV-transformed diseases.

A therapy for chronic hypergammaglobulinemeia evident in such diseasesas monoclonalgammopathy of undetermined significance (MGUS),Waldenstrom's disease, related idiopathic monoclonalgammopathies, andplasmacytomas.

A therapy for decreasing cellular proliferation of Large B-cellLymphomas.

A means of decreasing the involvement of B cells and Ig associated withChronic Myelogenous Leukemia.

An immunosuppressive agent(s).

IL17RLP polypeptides or polynucleotides of the invention, or antagonistsmay be used to modulate IgE concentrations in vitro or in vivo.

In another embodiment, administration of IL17RLP polypeptides orpolynucleotides of the invention, or antagonists thereof, may be used totreat, prevent, and/or diagnose IgE-mediated allergic reactionsincluding, but not limited to, asthma, rhinitis, and eczema.

An inhibitor of signaling pathways involving ERK1, COX2 and Cyclin D2which have been associated with IL17RLP-induced T or B cell activation.

The above-recited applications have uses in a wide variety of hosts.Such hosts include, but are not limited to, human, murine, rabbit, goat,guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken,goat, cow, sheep, dog, cat, non-human primate, and human. In specificembodiments, the host is a mouse, rabbit, goat, guinea pig, chicken,rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the hostis a mammal. In most preferred embodiments, the host is a human.

The agonists and antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as described above.

The antagonists may be employed for instance to inhibit IL17RLP-mediatedchemotaxis and activation of macrophages and their precursors, and ofneutrophils, basophils, T lymphocytes, B lymphocytes and some T-cellsubsets, e.g., activated and CD8 cytotoxic T cells and natural killercells, in certain auto-immune and chronic inflammatory and infectivediseases. Examples of auto-immune diseases include multiple sclerosis,and insulin-dependent diabetes. The antagonists may also be employed totreat, prevent, and/or diagnose infectious diseases including silicosis,sarcoidosis, idiopathic pulmonary fibrosis by preventing the recruitmentand activation of mononuclear phagocytes. They may also be employed totreat, prevent, and/or diagnose idiopathic hyper-eosinophilic syndromeby preventing eosinophil production and migration. Endotoxic shock mayalso be treated by the antagonists by preventing the migration ofmacrophages and their production of the IL17RLP polypeptides of thepresent invention. The antagonists may also be employed for treatingatherosclerosis, by preventing monocyte infiltration in the artery wall.The antagonists may also be employed to treat, prevent, and/or diagnosehistamine-mediated allergic reactions and immunological disordersincluding late phase allergic reactions, chronic urticaria, and atopicdermatitis by inhibiting chemokine-induced mast cell and basophildegranulation and release of histamine. IgE-mediated allergic reactionssuch as allergic asthma, rhinitis, and eczema may also be treated. Theantagonists may also be employed to treat, prevent, and/or diagnosechronic and acute inflammation by preventing the attraction of monocytesto a wound area. They may also be employed to regulate normal pulmonarymacrophage populations, since chronic and acute inflammatory pulmonarydiseases are associated with sequestration of mononuclear phagocytes inthe lung. Antagonists may also be employed to treat, prevent, and/ordiagnose rheumatoid arthritis by preventing the attraction of monocytesinto synovial fluid in the joints of patients. Monocyte influx andactivation plays a significant role in the pathogenesis of bothdegenerative and inflammatory arthropathies. The antagonists may beemployed to interfere with the deleterious cascades attributed primarilyto IL-1 and TNF, which prevents the biosynthesis of other inflammatorycytokines. In this way, the antagonists may be employed to preventinflammation. The antagonists may also be employed to inhibitprostaglandin-independent fever induced by IL17RLP. The antagonists mayalso be employed to treat, prevent, and/or diagnose cases of bone marrowfailure, for example, aplastic anemia and myelodysplastic syndrome. Theantagonists may also be employed to treat, prevent, and/or diagnoseasthma and allergy by preventing eosinophil accumulation in the lung.The antagonists may also be employed to treat, prevent, and/or diagnosesubepithelial basement membrane fibrosis which is a prominent feature ofthe asthmatic lung. The antagonists may also be employed to treat,prevent, and/or diagnose lymphomas (e.g., one or more of the extensive,but not limiting, list of lymphomas provided herein).

All of the above described applications as they may apply to veterinarymedicine. Moreover, all applications described herein may also apply toveterinary medicine.

Antibodies against IL17RLP may be employed to bind to and inhibitIL17RLP activity to treat, prevent, and/or diagnose ARDS, by preventinginfiltration of neutrophils into the lung after injury. The antagonistsand antagonists of the instant may be employed in a composition with apharmaceutically acceptable carrier, e.g., as described hereinafter.

IL17RLP polynucleotides or polypeptides of the invention and/or agonistsand/or antagonists thereof, are used to treat, prevent, and/or diagnosediseases and disorders of the pulmonary system (e.g., bronchi such as,for example, sinopulmonary and bronchial infections and conditionsassociated with such diseases and disorders and other respiratorydiseases and disorders. In specific embodiments, such diseases anddisorders include, but are not limited to, bronchial adenoma, bronchialasthma, pneumonia (such as, e.g., bronchial pneumonia, bronchopneumonia,and tuberculous bronchopneumonia), chronic obstructive pulmonary disease(COPD), bronchial polyps, bronchiectasia (such as, e.g., bronchiectasiasicca, cylindrical bronchiectasis, and saccular bronchiectasis),bronchiolar adenocarcinoma, bronchiolar carcinoma, bronchiolitis (suchas, e.g., exudative bronchiolitis, bronchiolitis fibrosa obliterans, andproliferative bronchiolitis), bronchiolo-alveolar carcinoma, bronchiticasthma, bronchitis (such as, e.g., asthmatic bronchitis, Castellani'sbronchitis, chronic bronchitis, croupous bronchitis, fibrinousbronchitis, hemorrhagic bronchitis, infectious avian bronchitis,obliterative bronchitis, plastic bronchitis, pseudomembranousbronchitis, putrid bronchitis, and verminous bronchitis), bronchocentricgranulomatosis, bronchoedema, bronchoesophageal fistula, bronchogeniccarcinoma, bronchogenic cyst, broncholithiasis, bronchomalacia,bronchomycosis (such as, e.g., bronchopulmonary aspergillosis),bronchopulmonary spirochetosis, hemorrhagic bronchitis, bronchorrhea,bronchospasm, bronchostaxis, bronchostenosis, Biot's respiration,bronchial respiration, Kussmaul respiration, Kussmaul-Kien respiration,respiratory acidosis, respiratory alkalosis, respiratory distresssyndrome of the newborn, respiratory insufficiency, respiratoryscleroma, respiratory syncytial virus, and the like.

In a specific embodiment, IL17RLP polynucleotides or polypeptides of theinvention and/or agonists and/or antagonists thereof, are used to treat,prevent, and/or diagnose chronic obstructive pulmonary disease (COPD).

In another embodiment, IL17RLP polynucleotides or polypeptides of theinvention and/or agonists and/or antagonists thereof, are used to treat,prevent, and/or diagnose fibroses and conditions associated withfibroses, such as, for example, but not limited to, cystic fibrosis(including such fibroses as cystic fibrosis of the pancreas,Clarke-Hadfield syndrome, fibrocystic disease of the pancreas,mucoviscidosis, and viscidosis), endomyocardial fibrosis, idiopathicretroperitoneal fibrosis, leptomeningeal fibrosis, mediastinal fibrosis,nodular subepidermal fibrosis, pericentral fibrosis, perimuscularfibrosis, pipestem fibrosis, replacement fibrosis, subadventitialfibrosis, and Symmers' clay pipestem fibrosis.

In another embodiment, IL17RLP polynucleotides or polypeptides of theinvention and/or agonists and/or antagonists thereof, are used to treat,prevent, and/or diagnose inner ear infection (such as, for example,otitis media), as well as other infections characterized by infectionwith Streptococcus pneumoniae and other pathogenic organisms.

IL17RLP polynucleotides or polypeptides of the invention and/or agonistsand/or antagonists thereof, are used to treat, prevent, and/or diagnosevarious immune system-related disorders and/or conditions associatedwith these disorders, in mammals, preferably humans. Many autoimmunedisorders result from inappropriate recognition of self as foreignmaterial by immune cells. This inappropriate recognition results in animmune response leading to the destruction of the host tissue.Therefore, the administration of IL17RLP polynucleotides or polypeptidesof the invention and/or agonists and/or antagonists thereof that caninhibit an immune response, particularly the proliferation,differentiation, or chemotaxis of T cells, may be an effective therapyin treating and/or preventing autoimmune disorders. Thus, in preferredembodiments, IL17RLP antagonists of the invention (e.g., polypeptidefragments of IL17RLP and anti-IL17RLP antibodies) are used to treat,prevent, and/or diagnose an autoimmune disorder.

Such autoimmune disorders include, but are not limited to, autoimmunediseases such as, for example, autoimmune hemolytic anemia, autoimmuneneonatal thrombocytopenia, autoimmunocytopenia, hemolytic anemia,antiphospholipid syndrome, dermatitis, allergic encephalomyelitis,glomerulonephritis, Multiple Sclerosis, Neuritis, Ophthalmia,Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome,Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulindependent diabetes mellitis, and autoimmune inflammatory eye disease.

Additional autoimmune disorders (that are highly probable) that may betreated, prevented, and/or diagnosed with the compositions of theinvention include, but are not limited to, autoimmune thyroiditis (i.e.,Hashimoto's thyroiditis) (often characterized, e.g., by cell-mediatedand humoral thyroid cytotoxicity), systemic lupus erhthematosus (oftencharacterized, e.g., by circulating and locally generated immunecomplexes), Goodpasture's syndrome (often characterized, e.g., byanti-basement membrane antibodies), Pemphigus (often characterized,e.g., by epidermal acantholytic antibodies), Receptor autoimmunitiessuch as, for example, (a) Graves' Disease (often characterized, e.g., byTSH receptor antibodies), (b) Myasthenia Gravis (often characterized,e.g., by acetylcholine receptor antibodies), and (c) insulin resistance(often characterized, e.g., by insulin receptor antibodies), autoimmunehemolytic anemia (often characterized, e.g., by phagocytosis ofantibody-sensitized RBCs), autoimmune thrombocytopenic purpura (oftencharacterized, e.g., by phagocytosis of antibody-sensitized platelets.

Additional autoimmune disorders (that are probable) that may be treated,prevented, and/or diagnosed with the compositions of the inventioninclude, but are not limited to, rheumatoid arthritis (oftencharacterized, e.g., by immune complexes in joints), scleroderma withanti-collagen antibodies (often characterized, e.g., by nucleolar andother nuclear antibodies), mixed connective tissue disease (oftencharacterized, e.g., by antibodies to extractable nuclear antigens(e.g., ribonucleoprotein)), polymyositis (often characterized, e.g., bynonhistone ANA), pernicious anemia (often characterized, e.g., byantiparietal cell, microsomes, and intrinsic factor antibodies),idiopathic Addison's disease (often characterized, e.g., by humoral andcell-mediated adrenal cytotoxicity, infertility (often characterized,e.g., by antispermatozoal antibodies), glomerulonephritis (oftencharacterized, e.g., by glomerular basement membrane antibodies orimmune complexes), bullous pemphigoid (often characterized, e.g., by IgGand complement in basement membrane), Sjogren's syndrome (oftencharacterized, e.g., by multiple tissue antibodies, and/or a specificnonhistone ANA (SS-B)), diabetes millitus (often characterized, e.g., bycell-mediated and humoral islet cell antibodies), and adrenergic drugresistance (including adrenergic drug resistance with asthma or cysticfibrosis) (often characterized, e.g., by beta-adrenergic receptorantibodies).

Additional autoimmune disorders (that are possible) that may be treated,prevented, and/or diagnosed with the compositions of the inventioninclude, but are not limited to, chronic active hepatitis (oftencharacterized, e.g., by smooth muscle antibodies), primary biliarycirrhosis (often characterized, e.g., by mitchondrial antibodies), otherendocrine gland failure (often characterized, e.g., by specific tissueantibodies in some cases), vitiligo (often characterized, e.g., bymelanocyte antibodies), vasculitis (often characterized, e.g., by Ig andcomplement in vessel walls and/or low serum complement), post-MI (oftencharacterized, e.g., by myocardial antibodies), cardiotomy syndrome(often characterized, e.g., by myocardial antibodies), urticaria (oftencharacterized, e.g., by IgG and IgM antibodies to IgE), atopicdermatitis (often characterized, e.g., by IgG and IgM antibodies toIgE), asthma (often characterized, e.g., by IgG and 10/1 antibodies toIgE), and many other inflammatory, granulamatous, degenerative, andatrophic disorders.

In a preferred embodiment, the autoimmune diseases and disorders and/orconditions associated with the diseases and disorders recited above aretreated, prevented, and/or diagnosed using anti-IL17RLP antibodiesand/or a soluble IL17RLP polypeptide of the invention (e.g., anextracellular domain of IL17RLP).

In a specific preferred embodiment, rheumatoid arthritis is treated,prevented, and/or diagnosed using anti-IL17RLP antibodies and/or asoluble IL17RLP polypeptide and/or other antagonist of the invention.

In a specific preferred embodiment, lupus is treated, prevented, and/ordiagnosed using anti-IL17RLP antibodies and/or a soluble IL17RLPpolypeptide and/or other antagonist of the invention.

In a specific preferred embodiment, nephritis associated with lupus istreated, prevented, and/or diagnosed using anti-IL17RLP antibodiesand/or a soluble IL17RLP polypeptide and/or other antagonist of theinvention.

In a specific embodiment, IL17RLP polynucleotides or polypeptides, orantagonists thereof (e.g., anti-IL17RLP antibodies) are used to treat orprevent systemic lupus erythramatosus and/or diseases, disorders orconditions associated therewith. Lupus-associated diseases, disorders,or conditions that may be treated or prevented with IL17RLPpolynucleotides or polypeptides, or antagonists of the invention,include, but are not limited to, hematologic disorders (e.g., hemolyticanemia, leukopenia, lymphopenia, and thrombocytopenia), immunologicdisorders (e.g., anti-DNA antibodies, and anti-Sm antibodies), rashes,photosensitivity, oral ulcers, arthritis, fever, fatigue, weight loss,serositis (e.g., pleuritus (pleuricy)), renal disorders (e.g.,nephritis), neurological disorders (e.g., seizures, peripheralneuropathy, CNS related disorders), gastroinstestinal disorders, Raynaudphenomenon, and pericarditis. In a preferred embodiment, the IL17RLPpolynucleotides or polypeptides, or antagonists thereof (e.g.,anti-IL17RLP antibodies) are used to treat or prevent renal disordersassociated with systemic lupus erythramatosus. In a most preferredembodiment, IL17RLP polynucleotides or polypeptides, or antagoniststhereof (e.g., anti-IL17RLP antibodies) are used to treat or preventnephritis associated with systemic lupus erythramatosus.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by IL17RLP polynucleotides or polypeptides of the inventionand/or agonists and/or antagonists thereof. Moreover, these moleculescan be used to treat, prevent, and/or diagnose anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility.

IL17RLP polynucleotides or polypeptides of the invention and/or agonistsand/or antagonists thereof, may also be used to treat, prevent, and/ordiagnose organ rejection or graft-versus-host disease (GVHD) and/orconditions associated therewith. Organ rejection occurs by host immunecell destruction of the transplanted tissue through an immune response.Similarly, an immune response is also involved in GVHD, but, in thiscase, the foreign transplanted immune cells destroy the host tissues.The administration of IL17RLP polynucleotides or polypeptides of theinvention and/or agonists and/or antagonists thereof, that inhibits animmune response, particularly the proliferation, differentiation, orchemotaxis of T-cells, may be an effective therapy in preventing organrejection or GVHD.

Similarly, IL17RLP polynucleotides or polypeptides of the inventionand/or agonists and/or antagonists thereof, may also be used to modulateinflammation. For example, IL17RLP polynucleotides or polypeptides ofthe invention and/or agonists and/or antagonists thereof, may inhibitthe proliferation and differentiation of cells involved in aninflammatory response. These molecules can be used to treat, prevent,and/or diagnose inflammatory conditions, both chronic and acuteconditions, including chronic prostatitis, granulomatous prostatitis andmalacoplakia, inflammation associated with infection (e.g., septicshock, sepsis, or systemic inflammatory response syndrome (SIRS)),ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL-1.)

In a specific embodiment, anti-IL17RLP antibodies of the invention areused to treat, prevent, modulate, detect, and/or diagnose inflammation.

In a specific embodiment, anti-IL17RLP antibodies of the invention areused to treat, prevent, modulate, detect, and/or diagnose inflamatorydisorders.

In another specific embodiment, anti-IL17RLP antibodies of the inventionare used to treat, prevent, modulate, detect, and/or diagnose allergyand/or hypersensitivity.

The TNF family ligands are known to be among the most pleiotropiccytokines, inducing a large number of cellular responses, includingcytotoxicity, anti-viral activity, immunoregulatory activities, and thetranscriptional regulation of several genes (D. V. Goeddel et al.,“Tumor Necrosis Factors: Gene Structure and Biological Activities,”Symp. Quant. Biol. 51:597-609 (1986), Cold Spring Harbor, B. Beutler andA. Cerami, Annu. Rev. Biochem. 57:505-518 (1988); L. J. Old, Sci. Am.258:59-75 (1988); W. Fiers, FEBS Lett. 285:199-224 (1991)). IL17RLPpolypeptides are believed to elicit a potent cellular response includingany genotypic, phenotypic, and/or morphologic change to the cell, cellline, tissue, tissue culture or patient. As indicated, such cellularresponses include not only normal physiological responses to IL17RLP,but also diseases associated with increased apoptosis or the inhibitionof apoptosis. Apoptosis-programmed cell death-is a physiologicalmechanism involved in the deletion of peripheral B and/or T lymphocytesof the immune system, and its disregulation can lead to a number ofdifferent pathogenic processes (J. C. Ameisen, AIDS 8:1197-1213 (1994);P. H. Krammer et al., Curr. Opin. Immunol. 6:279-289 (1994)).

Diseases associated with increased cell survival, or the inhibition ofapoptosis, include cancers (such as follicular lymphomas, carcinomaswith p53 mutations, and hormone-dependent tumors, including, but notlimited to, colon cancer, cardiac tumors, pancreatic cancer, melanoma,retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicularcancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma,endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as systemiclupus erythematosus and immune-related glomerulonephritis rheumatoidarthritis); viral infections (such as herpes viruses, pox viruses andadenoviruses); inflammation; graft vs. host disease; acute graftrejection and chronic graft rejection. Thus, in preferred embodimentsIL17RLP polynucleotides or polypeptides of the invention are used totreat, prevent, and/or diagnose autoimmune diseases and/or inhibit thegrowth, progression, and/or metastasis of cancers, including, but notlimited to, those cancers disclosed herein, such as, for example,lymphocytic leukemias (including, for example, MLL and chroniclymphocytic leukemia (CLL)) and follicular lymphomas. In anotherembodiment IL17RLP polynucleotides or polypeptides of the invention areused to activate, differentiate or proliferate cancerous cells or tissue(e.g., B cell lineage related cancers (e.g., CLL and MLL), lymphocyticleukemia, or lymphoma) and thereby render the cells more vulnerable tocancer therapy (e.g., chemotherapy or radiation therapy).

Moreover, in other embodiments, IL17RLP polynucleotides or polypeptidesof the invention are used to inhibit the growth, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Diseases associated with increased apoptosis include AIDS;neurodegenerative disorders (such as Alzheimer's disease, Parkinson'sdisease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellardegeneration); myelodysplastic syndromes (such as aplastic anemia),ischemic injury (such as that caused by myocardial infarction, strokeand reperfusion injury), toxin-induced liver disease (such as thatcaused by alcohol), septic shock, cachexia and anorexia. Thus, inpreferred embodiments IL17RLP polynucleotides or polypeptides of theinvention are used to treat, prevent, and/or diagnose the diseases anddisorders listed above.

In preferred embodiments, IL17RLP polypeptides of the invention inhibitthe growth of human histiocytic lymphoma U-937 cells in a dose-dependentmanner. In additional preferred embodiments, IL17RLP polypeptides of theinvention inhibit the growth of PC-3 cells, HT-29 cells, HeLa cells,MCF-7 cells, and A293 cells. In highly preferred embodiments, IL17RLPpolynucleotides or polypeptides of the invention are used to inhibitgrowth, progression, and/or metastasis of prostate cancer, colon cancer,cervical carcinoma, and breast carcinoma.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are useful in the diagnosis and treatment orprevention of a wide range of diseases and/or conditions. Such diseasesand conditions include, but are not limited to, cancer (e.g., immunecell related cancers, breast cancer, prostate cancer, ovarian cancer,follicular lymphoma, cancer associated with mutation or alteration ofp53, brain tumor, bladder cancer, uterocervical cancer, colon cancer,colorectal cancer, non-small cell carcinoma of the lung, small cellcarcinoma of the lung, stomach cancer, etc.), lymphoproliferativedisorders (e.g., lymphadenopathy), microbial (e.g., viral, bacterial,etc.) infection (e.g., HIV-1 infection, HIV-2 infection, herpesvirusinfection (including, but not limited to, HSV-1, HSV-2, CMV, VZV, HHV-7,EBV), adenovirus infection, poxvirus infection, human papilloma virusinfection, hepatitis infection (e.g., HAV, HBV, HCV, etc.), Helicobacterpylori infection, invasive Staphylococcia, etc), parasitic infection,nephritis, bone disease (e.g., osteoporosis), atherosclerosis, pain,cardiovascular disorders (e.g., neovascularization, hypovascularizationor reduced circulation (e.g., ischemic disease (e.g., myocardialinfarction, stroke, etc.)), AIDS, allergy, inflammation,neurodegenerative disease (e.g., Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, pigmentary retinitis, cerebellardegeneration, etc.), graft rejection (acute and chronic), graft vs. hostdisease, diseases due to osteomyelodysplasia (e.g., aplastic anemia,etc.), joint tissue destruction in rheumatism, liver disease (e.g.,acute and chronic hepatitis, liver injury, and cirrhosis), autoimmunedisease (e.g., multiple sclerosis, rheumatoid arthritis, systemic lupuserythematosus, immune complex glomerulonephritis, autoimmune diabetes,autoimmune thrombocytopenic purpura, Grave's disease, Hashimoto'sthyroiditis, etc.), cardiomyopathy (e.g., dilated cardiomyopathy),diabetes, diabetic complications (e.g., diabetic nephropathy, diabeticneuropathy, diabetic retinopathy), influenza, asthma, psoriasis,glomerulonephritis, septic shock, and ulcerative colitis.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are useful in promoting angiogenesis, woundhealing (e.g., wounds, burns, and bone fractures). Polynucleotidesand/or polypeptides of the invention and/or agonists and/or antagoniststhereof are also useful as an adjuvant to enhance immune responsivenessto specific antigen, anti-viral immune responses.

More generally, polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof are useful in regulating(i.e., elevating or reducing) immune response. For example,polynucleotides and/or polypeptides of the invention may be useful inpreparation or recovery from surgery, trauma, radiation therapy,chemotherapy, and transplantation, or may be used to boost immuneresponse and/or recovery in the elderly and immunocompromisedindividuals. Alternatively, polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof are useful asimmunosuppressive agents, for example in the treatment or prevention ofautoimmune disorders. In specific embodiments, polynucleotides and/orpolypeptides of the invention are used to treat or prevent chronicinflammatory, allergic or autoimmune conditions, such as those describedherein or are otherwise known in the art.

Formulations

The IL17RLP polypeptide composition will be formulated and dosed in afashion consistent with good medical practice, taking into account theclinical condition of the individual patient (especially the sideeffects of treatment with IL17RLP polypeptide alone), the site ofdelivery of the IL17RLP polypeptide composition, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” of IL17RLP polypeptidefor purposes herein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofIL17RLP polypeptide administered parenterally per dose will be in therange of about 1 μg/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the IL17RLP polypeptide is typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by IL-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed. The length of treatment needed toobserve changes and the interval following treatment for responses tooccur appears to vary depending on the desired effect.

Effective dosages of the compositions of the present invention to beadministered may be determined through procedures well known to those inthe art which address such parameters as biological half-life,bioavailability, and toxicity. Such determination is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

Bioexposure of an organism to IL17RLP polypeptide during therapy mayalso play an important role in determining a therapeutically and/orpharmacologically effective dosing regime. Variations of dosing such asrepeated administrations of a relatively low dose of IL17RLP polypeptidefor a relatively long period of time may have an effect which istherapeutically and/or pharmacologically distinguishable from thatachieved with repeated administrations of a relatively high dose ofIL17RLP for a relatively short period of time.

Using the equivalent surface area dosage conversion factors supplied byFreireich, E. J., et al. (Cancer Chemotherapy Reports 50(4):219-44(1966)), one of ordinary skill in the art is able to convenientlyconvert data obtained from the use of IL17RLP in a given experimentalsystem into an accurate estimation of a pharmaceutically effectiveamount of IL17RLP polypeptide to be administered per dose in anotherexperimental system. Experimental data obtained through theadministration of IL17RLP in mice may converted through the conversionfactors supplied by Freireich, et al., to accurate estimates ofpharmaceutically effective doses of IL17RLP in rat, monkey, dog, andhuman. The following conversion table (Table III) is a summary of thedata provided by Freireich, et al. Table III gives approximate factorsfor converting doses expressed in terms of mg/kg from one species to anequivalent surface area dose expressed as mg/kg in another speciestabulated.

TABLE III Equivalent Surface Area Dosage Conversion Factors. TO DogMouse Rat Monkey (8 Human FROM (20 g) (150 g) (3.5 kg) kg) (60 kg) Mouse1 1/2 1/4 1/6  1/12 Rat 2 1 1/2 1/4 1/7 Monkey 4 2 1 3/5 1/3 Dog 6 4 5/31 1/2 Human 12 7 3 2 1

Thus, for example, using the conversion factors provided in Table III, adose of 50 mg/kg in the mouse converts to an appropriate dose of 123mg/kg in the monkey because (50 mg/kg)×(¼)=12.5 mg/kg. As an additionalexample, doses of 0.02, 0.08, 0.8, 2, and 8 mg/kg in the mouse equate toeffect doses of 1.667 micrograms/kg, 6.67 micrograms/kg, 66.7micrograms/kg, 166.7 micrograms/kg, and 0.667 mg/kg, respectively, inthe human.

Pharmaceutical compositions containing the IL17RLP of the invention maybe administered orally, rectally, parenterally, intracistemally,intravaginally, intraperitoneally, topically (as by powders, ointments,drops or transdermal patch), bucally, or as an oral or nasal spray. By“pharmaceutically acceptable carrier” is meant a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. In a specific embodiment,“pharmaceutically acceptable” means approved by a regulatory agency ofthe federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly humans. Nonlimiting examples of suitable pharmaceuticalcarriers according to this embodiment are provided in “Remington'sPharmaceutical Sciences” by E. W. Martin, and include sterile liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can be employed asliquid carriers, particularly for injectable solutions. The composition,if desired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like.

The term “parenteral” as used herein refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion.

In a preferred embodiment, IL17RLP compositions of the invention(including polypeptides, polynucleotides, and antibodies, and agonistsand/or antagonists thereof) are administered subcutaneously.

In another preferred embodiment, IL17RLP compositions of the invention(including polypeptides, polynucleotides, and antibodies, and agonistsand/or antagonists thereof) are administered intravenously.

The IL17RLP polypeptide is also suitably administered bysustained-release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or mirocapsules, suitable hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, and sparingly soluble derivatives (such as, forexample, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, U., et al, Biopolymers 22:547-556(1983)), poly (2-hydroxyethyl methacrylate; Langer, R., et al, J. BiomedMater. Res. 15:167-277 (1981), and Langer, R., Chem. Tech 12:98-105(1982)), ethylene vinyl acetate (Langer, R., et al, Id) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-release IL17RLPpolypeptide compositions also include liposomally entrapped IL17RLPpolypeptide. Liposomes containing IL17RLP polypeptide are prepared bymethods known in the art (DE 3,218,121; Epstein, et al, Proc. Natl Acad.Sci. (USA) 82:3688-3692 (1985); Hwang, et al, Proc. Natl. Acad Sci (USA)77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324). Ordinarily, the liposomes are of the small(about 200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. percent cholesterol, the selected proportionbeing adjusted for the optimal IL17RLP polypeptide therapy.

In another embodiment sustained release compositions of the inventioninclude crystal formulations known in the art.

In yet an additional embodiment, the compositions of the invention aredelivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref.Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, the IL17RLPpolypeptide is formulated generally by mixing it at the desired degreeof purity, in a unit dosage injectable form (solution, suspension, oremulsion), with a pharmaceutically acceptable carrier, i.e., one that isnon-toxic to recipients at the dosages and concentrations employed andis compatible with other ingredients of the formulation. For example,the formulation preferably does not include oxidizing agents and othercompounds that are known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the IL17RLPpolypeptide uniformly and intimately with liquid carriers or finelydivided solid carriers or both. Then, if necessary, the product isshaped into the desired formulation. Preferably the carrier is aparenteral carrier, more preferably a solution that is isotonic with theblood of the recipient. Examples of such carrier vehicles include water,saline, Ringer's solution, and dextrose solution. Non-aqueous vehiclessuch as fixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The IL17RLP polypeptide is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of IL17RLP polypeptide salts.

IL17RLP polypeptide to be used for therapeutic administration must besterile. Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeutic IL17RLPpolypeptide compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

IL17RLP polypeptide ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous IL17RLP polypeptide solution, andthe resulting mixture is lyophilized. The infusion solution is preparedby reconstituting the lyophilized IL17RLP polypeptide usingbacteriostatic water-for-injection (WFI).

Alternatively, IL17RLP polypeptide is stored in single dose containersin lyophilized form. The infusion selection is reconstituted using asterile carrier for injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides of the present invention may be employed in conjunctionwith other therapeutic compounds.

The compositions of the invention may be administered alone or incombination with other adjuvants. Adjuvants that may be administeredwith the compositions of the invention include, but are not limited to,alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(GENENTECH™, Inc.), BCG, and MPL. In a specific embodiment, compositionsof the invention are administered in combination with alum. In anotherspecific embodiment, compositions of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe compositions of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccinesthat may be administered with the compositions of the invention include,but are not limited to, vaccines directed toward protection against MMR(measles, mumps, rubella), polio, varicella, tetanus/diptheria,hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough,pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever,Japanese encephalitis, poliomyelitis, rabies, typhoid fever, andpertussis, and/or PNEUMOVAX-23™. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In another specific embodiment, compositions of the invention are usedin combination with PNEUMOVAX-23″ to treat, prevent, and/or diagnoseinfection and/or any disease, disorder, and/or condition associatedtherewith. In one embodiment, compositions of the invention are used incombination with PNEUMOVAX-23™ to treat, prevent, and/or diagnose anyGram positive bacterial infection and/or any disease, disorder, and/orcondition associated therewith. In another embodiment, compositions ofthe invention are used in combination with PNEUMOVAX-23™ to treat,prevent, and/or diagnose infection and/or any disease, disorder, and/orcondition associated with one or more members of the genus Enterococcusand/or the genus Streptococcus. In another embodiment, compositions ofthe invention are used in any combination with PNEUMOVAX-23″ to treat,prevent, and/or diagnose infection and/or any disease, disorder, and/orcondition associated with one or more members of the Group Bstreptococci. In another embodiment, compositions of the invention areused in combination with PNEUMOVAX-23™ to treat, prevent, and/ordiagnose infection and/or any disease, disorder, and/or conditionassociated with Streptococcus pneumoniae.

The compositions of the invention may be administered alone or incombination with other therapeutic agents, including but not limited to,chemotherapeutic agents, antibiotics, antivirals, steroidal andnon-steroidal anti-inflammatories, conventional immunotherapeutic agentsand cytokines. Combinations may be administered either concomitantly,e.g., as an admixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In one embodiment, the compositions of the invention are administered incombination with one or more members of the TNF family. TNF, TNF-relatedor TNF-like molecules that may be administered with the compositions ofthe invention include, but are not limited to, soluble forms ofTNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta(found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L,CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International PublicationNo. WO 96/14328), AIM-I (International Publication No. WO 97/33899),AIM-II (International Publication No. WO 97/34911), APRIL (I. Exp. Med.188(6):1185-1190), endokine-alpha (International Publication No. WO98/07880), TR6 (International Publication No. WO 98/30694), OPG, andneutrokine-alpha (International Publication No. WO 98/18921, OX40, andnerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3(International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TR6 (International Publication No. WO 98/30694), TR7(International Publication No. WO 98/41629), TRANK, TR9 (InternationalPublication No. WO 98/56892), TR10 (International Publication No. WO98/54202), 312C2 (International Publication No. WO 98/06842), and TR12.

In a preferred embodiment, the compositions of the invention areadministered in combination with CD40 ligand (CD40L), a soluble form ofCD40L (e.g., AVREND™), bioloigically active fragments, variants, orderivatives of CD40L, anti-CD40L antibodies (e.g., agonistic orantagonistic antibodies), and/or anti-CD40 antibodies (e.g, agonistic orantagonistic antibodies).

In certain embodiments, compositions of the invention are administeredin combination with antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the compositionsof the invention, include, but are not limited to, RETROVIR™(zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC),ZERIT™ (stavudine/d4T), EPEVIR™ (lamivudine/3TC), and COMBIVIR™(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, CRIXIVAN™ (indinavir),NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith compositions of the invention to treat, prevent, and/or diagnoseAIDS and/or to treat, prevent, and/or diagnose HIV infection.

In other embodiments, compositions of the invention may be administeredin combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe compositions of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, compositions of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat, prevent, and/or diagnose anopportunistic Pneumocystis carinii pneumonia infection. In anotherspecific embodiment, compositions of the invention are used in anycombination with ESONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/orETHAMBUTOL™ to prophylactically treat, prevent, and/or diagnose anopportunistic Mycobacterium avium complex infection. In another specificembodiment, compositions of the invention are used in any combinationwith RIFABUTIN™, CLARITHROMYCIN™, and/or AZITHROMYCIN™ toprophylactically treat, prevent, and/or diagnose an opportunisticMycobacterium tuberculosis infection. In another specific embodiment,compositions of the invention are used in any combination withGANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylactically treat,prevent, and/or diagnose an opportunistic cytomegalovirus infection. Inanother specific embodiment, compositions of the invention are used inany combination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™to prophylactically treat, prevent, and/or diagnose an opportunisticfungal infection. In another specific embodiment, compositions of theinvention are used in any combination with ACYCLOVIR™ and/orFAMCICOLVIR™ to prophylactically treat, prevent, and/or diagnose anopportunistic herpes simplex virus type I and/or type II infection. Inanother specific embodiment, compositions of the invention are used inany combination with PYRIMETHAMINE™ and/or LEUCOVORIN™ toprophylactically treat, prevent, and/or diagnose an opportunisticToxoplasma gondii infection. In another specific embodiment,compositions of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat, prevent, and/ordiagnose an opportunistic bacterial infection.

In a further embodiment, the compositions of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the compositions of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, amoxicillin, aminoglycosides, beta-lactam(glycopeptide), beta-lactamases, Clindamycin, chloramphenicol,cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin,fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim,trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

In specific embodiments, compositions of the invention are administeredin combination with immunosuppressants. Immunosuppressants preparationsthat may be administered with the compositions of the invention include,but are not limited to, ORTHOCLONE™ (0KT3), SANDIMMUNE™/NEORAL™/SANGDYA™(cyclosporin), PROGRAF™ (tacrolimus), CELLCEPT™ (mycophenolate),Azathioprine, glucorticosteroids, and RAPAMUNE™ (sirolimus). In aspecific embodiment, immunosuppressants may be used to prevent rejectionof organ or bone marrow transplantation.

In an additional embodiment, compositions of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the compositions of the invention include, but notlimited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, andGAMIMUNE™. In a specific embodiment, compositions of the invention areadministered in combination with intravenous immune globulinpreparations in transplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the compositionsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compostions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the compositions of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, compositions of the invention are administeredin combination with CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone) or any combination of the components of CHOP. In anotherembodiment, compositions of the invention are administered incombination with Rituximab. In a further embodiment, compositions of theinvention are administered with Rituxmab and CHOP, or Rituxmab and anycombination of the components of CHOP.

In an additional embodiment, the compositions of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, GM-CSF, G-CSF, IL2, IL3, IL4, 115, IL6, IL7, IL 10, IL12,IL13, IL15, IL17, IL19, IL20, IL21, IL22, anti-CD40, CD40L, IFN-alpha,IFN-beta, IFN-gamma, TNF-alpha, and TNF-beta. In another embodiment,compositions of the invention may be administered with any interleukin,including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, and IL-22.

In an additional embodiment, the compositions of the invention areadministered with a chemokine. In another embodiment, the compositionsof the invention are administered with chemokine beta-8, chemokinebeta-1, and/or macrophage inflammatory protein-4. In a preferredembodiment, the compositions of the invention are administered withchemokine beta-8.

In an additional embodiment, the compositions of the invention areadministered in combination with an IL-4 antagonist. IL-4 antagoniststhat may be administered with the compositions of the invention include,but are not limited to: soluble IL-4 receptor polypeptides, multimericforms of soluble IL-4 receptor polypeptides; anti-IL-4 receptorantibodies that bind the IL-4 receptor without transducing thebiological signal elicited by IL-4, anti-IL4 antibodies that blockbinding of IL-4 to one or more IL-4 receptors, and muteins of IL-4 thatbind IL-4 receptors but do not transduce the biological signal elicitedby IL-4. Preferably, the antibodies employed according to this methodare monoclonal antibodies (including antibody fragments, such as, forexample, those described herein).

In an additional embodiment, the compositions of the invention areadministered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with thecompositions of the invention include, but are not limited to, LEUKINE™(SARGRAMOSTIM™) and NEUPOGEN™ (FELGRASTIM™).

In an additional embodiment, the compositions of the invention areadministered in combination with fibroblast growth factors. Fibroblastgrowth factors that may be administered with the compositions of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

Additionally, the compositions of the invention may be administeredalone or in combination with other therapeutic regimens, including butnot limited to, radiation therapy. Such combinatorial therapy may beadministered sequentially and/or concomitantly.

Agonists and Antagonists—Assays and Molecules

The invention also provides a method of screening compounds to identifythose which enhance or block the action of IL17RLP on cells, such as itsinteraction with IL17RLP-binding molecules such as ligand molecules. Anagonist is a compound which increases the natural biological functionsof IL17RLP or which functions in a manner similar to IL17RLP, whileantagonists decrease or eliminate such functions.

In another aspect of this embodiment the invention provides a method foridentifying a ligand protein which binds specifically to a IL17RLPpolypeptide. For example, a cellular compartment, such as a membrane ora preparation thereof, may be prepared from a cell that expresses amolecule that binds IL17RLP. The preparation is incubated with labeledIL17RLP and complexes of IL17RLP bound to the ligand or other bindingprotein are isolated and characterized according to routine methodsknown in the art. Alternatively, the IL17RLP polypeptide may be bound toa solid support so that binding molecules solubilized from cells arebound to the column and then eluted and characterized according toroutine methods.

In the assay of the invention for agonists or antagonists, a cellularcompartment, such as a membrane or a preparation thereof, may beprepared from a cell that expresses a molecule that binds IL17RLP, suchas a molecule of a signaling or regulatory pathway modulated by IL17RLP.The preparation is incubated with labeled IL17RLP in the absence or thepresence of a candidate molecule which may be a IL17RLP agonist orantagonist. The ability of the candidate molecule to bind the bindingmolecule is reflected in decreased binding of the labeled ligand.Molecules which bind gratuitously, i.e., without inducing the effects ofIL17RLP on binding the IL17RLP binding molecule, are most likely to begood antagonists. Molecules that bind well and elicit effects that arethe same as or closely related to IL17RLP are agonists.

IL17RLP-like effects of potential agonists and antagonists may bymeasured, for instance, by determining activity of a second messengersystem following interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that ofIL17RLP or molecules that elicit the same effects as IL17RLP. Secondmessenger systems that may be useful in this regard include but are notlimited to AMP guanylate cyclase, ion channel or phosphoinositidehydrolysis second messenger systems.

Another example of an assay for IL17RLP antagonists is a competitiveassay that combines an IL17RLP ligand and a potential antagonist withmembrane-bound IL17RLP receptor molecules or recombinant IL17RLPreceptor molecules under appropriate conditions for a competitiveinhibition assay. The IL17RLP ligand can be labeled, such as byradioactivity, such that the number of IL17RLP ligand molecules bound toa receptor molecule can be determined accurately to assess theeffectiveness of the potential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, without inducing IL17RLP-induced activities, therebypreventing the action of IL17RLP by excluding the IL17RLP ligand frombinding.

Other potential antagonists include antisense molecules. Antisensetechnology can be used to control gene expression through antisense DNAor RNA or through triple-helix formation. Antisense techniques arediscussed in a number of studies (for example, Okano, J. Neurochem.56:560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression.” CRC Press, Boca Raton, Fla. (1988)). Triple helix formationis discussed in a number of studies, as well (for instance, Lee, et al.,Nucleic Acids Research 6:3073 (1979); Cooney, et al., Science 241:456(1988); Dervan, et al., Science 251:1360 (1991)). The methods are basedon binding of a polynucleotide to a complementary DNA or RNA. Forexample, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of IL17RLP. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into IL17RLP polypeptide. The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of IL17RLPprotein. The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of IL17RLP.

In one embodiment, the IL17RLP antisense nucleic acid of the inventionis produced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the IL17RLP antisense nucleic acid. Such avector can remain episomal or become chromosomally integrated, as longas it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others know inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding IL17RLP, or fragments thereof, canbe by any promoter known in the art to act in vertebrate, preferablyhuman cells. Such promoters can be inducible or constitutive. Suchpromoters include, but are not limited to, the SV40 early promoterregion (Bernoist and Chambon, Nature 29:304-310 (1981), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al., Cell 22:787-797 (1980), the herpes thymidine promoter (Wagner etal., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatorysequences of the metallothionein gene (Brinster, et al., Nature296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of an IL17RLPgene. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA,”referred to herein, means a sequence having sufficient complementarilyto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded IL17RLP antisense nucleic acids, a single strandof the duplex DNA may thus be tested, or triplex formation may beassayed. The ability to hybridize will depend on both the degree ofcomplementarity and the length of the antisense nucleic acid Generally,the larger the hybridizing nucleic acid, the more base mismatches with aIL17RLP RNA it may contain and still form a stable duplex (or triplex asthe case may be). One skilled in the art can ascertain a tolerabledegree of mismatch by use of standard procedures to determine themelting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333-335. Thus, oligonucleotides complementary to either the 5′- or3′-non-translated, non-coding regions of IL17RLP could be used in anantisense approach to inhibit translation of endogenous IL17RLP mRNA.Oligonucleotides complementary to the 5′ untranslated region of the mRNAshould include the complement of the AUG start codon. Antisenseoligonucleotides complementary to mRNA coding regions are less efficientinhibitors of translation but could be used in accordance with theinvention. Whether designed to hybridize to the 5′-, 3′- or codingregion of IL17RLP mRNA, antisense nucleic acids should be at least sixnucleotides in length, and are preferably oligonucleotides ranging from6 to about 50 nucleotides in length. In specific aspects theoligonucleotide is at least 10 nucleotides, at least 17 nucleotides, atleast 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556; Lemaitre et al., Proc. Natl. Acad. Sci. 84:648-652(1987); PCT Publication No. WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., BioTechniques 6:958-976 (1988)) or intercalatingagents. (See, e.g., Zon, Pharm. Res. 5:539-549 (1988)). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is analpha-anomeric oligonucleotide. An alpha-anomeric oligonucleotide formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual beta-units, the strands run parallel to each other(Gautier et al., Nucl. Acids Res. 15:6625-6641 (1987)). Theoligonucleotide is a 2-0-methylribonucleotide (Inoue et al., Nucl. AcidsRes. 15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al.,FEBS Lett. 215:327-330 (1997)).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (Nucl. Acids Res. 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A.85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to the IL17RLP coding regionsequence could be used, those complementary to the transcribeduntranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy IL17RLP mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA have thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, Nature 334:585-591 (1988).There are numerous potential hammerhead ribozyme cleavage sites withinthe nucleotide sequence of IL17RLP. Preferably, the ribozyme isengineered so that the cleavage recognition site is located near the 5′end of the IL17RLP mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express IL17RLPin vivo. DNA constructs encoding the ribozyme may be introduced into thecell in the same manner as described above for the introduction ofantisense encoding DNA. A preferred method of delivery involves using aDNA construct “encoding” the ribozyme under the control of a strongconstitutive promoter, such as, for example, pol III or pol II promoter,so that transfected cells will produce sufficient quantities of theribozyme to destroy endogenous IL17RLP messages and inhibit translation.Since ribozymes unlike antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

Endogenous gene expression can also be reduced by inactivating or“knocking out” the IL17RLP gene and/or its promoter using targetedhomologous recombination. (E.g., see Smithies et al., Nature 317:230-234(1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart. The contents of each of the documents recited in this paragraph isherein incorporated by reference in its entirety.

In other embodiments, antagonists according to the present inventioninclude soluble forms of IL17RLP (e.g., the extracellular domain ofIL17RLP). Such soluble forms of the IL17RLP, which may be naturallyoccurring or synthetic, antagonize IL17RLP-mediated signaling bycompeting with native IL17RLP for binding to IL17RLP ligands (e.g., IL20(See, International Application No. US98/14609)), and/or by forming amultimer that may or may not be capable of binding the ligand, but whichis incapable of inducing signal transduction. Preferably, theseantagonists inhibit IL17RLP-mediated stimulation of lymphocyte (e.g., Tor B cell) proliferation, differentiation, and/or activation.Antagonists of the present invention also include, for example,anti-IL17RLP antibodies IL17RLP-Fc fusion proteins.

Antagonists of the present invention also include antibodies specificfor IL17RLP polypeptides of the invention. Antibodies according to thepresent invention may be prepared by any of a variety of standardmethods using IL17RLP immunogens of the present invention. As indicated,such IL17RLP immunogens include the complete IL17RLP polypeptidesdepicted in SEQ ED NO:2 and SEQ ED NO:18 (which may or may not includethe leader sequence) and IL17RLP polypeptide fragments comprising, forexample, the extracellular domain.

Polyclonal and monoclonal antibody agonists or antagonists according tothe present invention can be raised according to the methods disclosedin Tartaglia and Goeddel, J. Biol. Chem. 267(7):4304-4307 (1992));Tartaglia et al., Cell 73:213-216 (1993)), and PCT Application WO94/09137 and are preferably specific to (i.e., bind uniquely topolypeptides of the invention having the amino acid sequence of SEQ IDNO:2. The term “antibody” (Ab) or “monoclonal antibody” (mAb) as usedherein is meant to include intact molecules as well as fragments thereof(such as, for example, Fab and F(ab′) fragments) which are capable ofbinding an antigen. Fab, Fab′ and F(ab′) fragments lack the Fc fragmentintact antibody, clear more rapidly from the circulation, and may haveless non-specific tissue binding of an intact antibody (Wahl et al., J.Nucl. Med, 24:316-325 (1983)).

In a preferred method, antibodies according to the present invention aremAbs. Such mAbs can be prepared using hybridoma technology (Kohler andMillstein, Nature 256:495-497 (1975) and U.S. Pat. No. 4,376,110; Harlowet al., Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988; Monoclonal Antibodies andHybridomas: A New Dimension in Biological Analyses, Plenum Press, NewYork, N.Y., 1980; Campbell, “Monoclonal Antibody Technology,” In:Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13(Burdon et al., eds.), Elsevier, Amsterdam (1984)).

Proteins and other compounds which bind the IL17RLP domains are alsocandidate agonists and antagonists according to the present invention.Such binding compounds can be “captured” using the yeast two-hybridsystem (Fields and Song, Nature 340:245-246 (1989)). A modified versionof the yeast two-hybrid system has been described by Roger Brent and hiscolleagues (Gyuris, Cell 75:791-803 (1993); Zervos et al., Cell72:223-232 (1993)). Preferably, the yeast two-hybrid system is usedaccording to the present invention to capture compounds which bind tothe ligand binding domain, extracellular, intracellular, andtransmembrane domains of IL17RLP. Such compounds are good candidateagonists and antagonists of the present invention.

For example, using the two-hybrid assay described above, theextracellular or intracellular domain of the IL17RLP receptor, or aportion thereof, may be used to identify cellular proteins whichinteract with the IL17RLP receptor in vivo. Such an assay may also beused to identify ligands with potential agonistic or antagonisticactivity of IL17RLP receptor function. This screening assay haspreviously been used to identify protein which interact with thecytoplasmic domain of the murine TNF-RII and led to the identificationof two receptor associated proteins. Rothe et al., Cell 78:681 (1994).Such proteins and amino acid sequences which bind to the cytoplasmicdomain of the IL17RLP are good candidate agonists and/or antagonists ofthe present invention.

Other screening techniques include the use of cells which express thepolypeptide of the present invention (for example, transfected CHOcells) in a system which measures extracellular changes caused byreceptor activation, for example, as described in Science, 246:181-296(1989). In another example, potential agonists or antagonists may becontacted with a cell which expresses the polypeptide of the presentinvention and a second messenger response, e.g., signal transduction maybe measured to determine whether the potential antagonist or agonist iseffective.

Agonists according to the present invention include naturally occurringand synthetic compounds such as, for example, TNF family ligand peptidefragments, transforming growth factor, neurotransmitters (such asglutamate, dopamine, N-methyl-D-aspartate), tumor suppressors (p53),cytolytic T cells and antimetabolites. Preferred agonists includechemotherapeutic drugs such as, for example, cisplatin, doxorubicin,bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate andvincristine. Others include ethanol and alpha- and/or beta-amyloidpeptide. (Science 267:1457-1458 (1995)).

Preferred agonists are fragments of IL7RLP polypeptides of the inventionwhich stimulate lymphocyte (e.g., T or B cell) proliferation,differentiation and/or activation. Further preferred agonists includepolyclonal and monoclonal antibodies raised against the IL17RLPpolypeptides of the invention, or a fragment thereof. Such agonistantibodies raised against a TNF-family receptor are disclosed inTartaglia et al., Proc. Natl. Acad Sci. USA 88:9292-9296 (1991); andTartaglia et al., J. Biol. Chem. 267:4304-4307 (1992). See, also, PCTApplication WO 94/09137.

In an additional embodiment, immunoregulatory molecules such as, forexample, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha, may be used as agonists ofIL17RLP polypeptides of the invention which stimulate lymphocyte (e.g.,T or B cell) proliferation, differentiation and/or activation. In aspecific embodiment, IL4 and/or IL10 are used to enhance theIL17RLP-mediated proliferation of T or B cells.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implantedin the body, e.g., genetically engineered fibroblasts can be implantedas part of a skin graft; genetically engineered endothelial cells can beimplanted as part of a lymphatic or vascular graft. (See, for example,Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S.Pat. No. 5,460,959 each of which is incorporated by reference herein inits entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

In yet another embodiment of the invention, the activity of IL17RLPpolypeptide can be reduced using a “dominant negative.” To this end,constructs which encode defective IL17RLP polypeptide, such as, forexample, mutants lacking all or a portion of any conserved domains, canbe used in gene therapy approaches to diminish the activity of IL17RLPon appropriate target cells.

The agonists and antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as described above.

The antagonists may be employed for instance to inhibit the activationof macrophages and their precursors, and of neutrophils, basophils, Blymphocytes and some T-cell subsets, e.g., activated and CD8 cytotoxic Tcells and natural killer cells, in certain auto-immune and chronicinflammatory and infective diseases. Examples of auto-immune diseasesinclude multiple sclerosis, and insulin-dependent diabetes. Theantagonists may also be employed to treat infectious diseases includingsilicosis, sarcoidosis, idiopathic pulmonary fibrosis by preventing theactivation of mononuclear phagocytes. They may also be employed to treatidiopathic hyper-eosinophilic syndrome by preventing eosinophilproduction. Antagonists may also be employed to treat rheumatoidarthritis by preventing the activation of monocytes in the synovialfluid in the joints of patients. Monocyte activation plays a significantrole in the pathogenesis of both degenerative and inflammatoryarthropathies. The antagonists may be employed to interfere with thedeleterious cascades attributed primarily to IL-1 and TNF, whichprevents the biosynthesis of other inflammatory cytokines. In this way,the antagonists may be employed to prevent inflammation. Antibodiesagainst IL17RLP may be employed to bind to and inhibit IL17RLP activityto treat such conditions described above. Any of the above antagonistsmay be employed in a composition with a pharmaceutically acceptablecarrier, e.g., as hereinafter described.

Gene Mapping (Chromosome Assays)

The nucleic acid molecules of the present invention are also valuablefor chromosome identification. The sequence is specifically targeted toand can hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a IL17RLP protein gene. Thiscan be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAthen is used for in situ chromosome mapping using well known techniquesfor this purpose.

In addition, in some cases, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp) from the cDNA. Computeranalysis of the 3′ untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Fluorescence in situ hybridization (“FISH”) of a cDNA cloneto a metaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp (for a review of this technique,see Verma, et al. Human Chromosomes: A Manual Of Basic Techniques,Pergamon Press, New York (1988)).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, on the WorldWide Web (McKusick, V. Mendelian Inheritance In Man, available on-linethrough Johns Hopkins University, Welch Medical Library). Therelationship between genes and diseases that have been mapped to thesame chromosomal region are then identified through linkage analysis(coinheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Utilizing the techniques described above, the chromosomal location ofIL17RLP was determined with high confidence using a combination ofsomatic cell hybrids and radiation hybrids to chromosome position3p21.1. It is noted that several chemokine receptors and trypsininhibitors have been mapped in the 3p21.1, 3p21.2, and 3p21 regions.

Binding Peptides and Other Molecules

The invention also encompasses screening methods for identifyingpolypeptides and nonpolypeptides that bind IL17RLP polypeptides, and theIL17RLP binding molecules identified thereby. These binding moleculesare useful, for example, as agonists and antagonists of the IL17RLPpolypeptides. Such agonists and antagonists can be used, in accordancewith the invention, in the therapeutic embodiments described in detail,below.

This method comprises the steps of:

contacting IL17RLP polypeptides or IL17RLP-like polypeptides with aplurality of molecules; and

identifying a molecule that binds the IL17RLP polypeptides orIL17RLP-like polypeptides.

The step of contacting the IL17RLP polypeptides or IL17RLP-likepolypeptides with the plurality of molecules may be effected in a numberof ways. For example, one may contemplate immobilizing the IL17RLPpolypeptides or IL17RLP-like polypeptides on a solid support andbringing a solution of the plurality of molecules in contact with theimmobilized IL17RLP polypeptides or IL17RLP-like polypeptides. Such aprocedure would be akin to an affinity chromatographic process, with theaffinity matrix being comprised of the immobilized IL17RLP polypeptidesor IL17RLP-like polypeptides. The molecules having a selective affinityfor the IL17RLP polypeptides or IL17RLP-like polypeptides can then bepurified by affinity selection. The nature of the solid support, processfor attachment of the IL17RLP polypeptides or IL17RLP-like polypeptidesto the solid support, solvent, and conditions of the affinity isolationor selection are largely conventional and well known to those ofordinary skill in the art.

Alternatively, one may also separate a plurality of polypeptides intosubstantially separate fractions comprising a subset of or individualpolypeptides. For instance, one can separate the plurality ofpolypeptides by gel electrophoresis, column chromatography, or likemethod known to those of ordinary skill for the separation ofpolypeptides. The individual polypeptides can also be produced by atransformed host cell in such a way as to be expressed on or about itsouter surface (e.g., a recombinant phage). Individual isolates can thenbe “probed” by the IL17RLP polypeptides or IL17RLP-like polypeptides,optionally in the presence of an inducer should one be required forexpression, to determine if any selective affinity interaction takesplace between the IL17RLP polypeptides or IL17RLP-like polypeptides andthe individual clone. Prior to contacting the IL17RLP polypeptides orIL17RLP-like polypeptides with each fraction comprising individualpolypeptides, the polypeptides could first be transferred to a solidsupport for additional convenience. Such a solid support may simply be apiece of filter membrane, such as one made of nitrocellulose or nylon.In this manner, positive clones could be identified from a collection oftransformed host cells of an expression library, which harbor a DNAconstruct encoding a polypeptide having a selective affinity for IL17RLPpolypeptides or IL17RLP-like polypeptides. Furthermore, the amino acidsequence of the polypeptide having a selective affinity for the IL17RLPpolypeptides or IL17RLP-like polypeptides can be determined directly byconventional means or the coding sequence of the DNA encoding thepolypeptide can frequently be determined more conveniently. The primarysequence can then be deduced from the corresponding DNA sequence. If theamino acid sequence is to be determined from the polypeptide itself, onemay use microsequencing techniques. The sequencing technique may includemass spectroscopy.

In certain situations, it may be desirable to wash away any unboundIL17RLP polypeptides or IL17RLP-like polypeptides, or alternatively,unbound polypeptides, from a mixture of the IL17RLP polypeptides orIL17RLP-like polypeptides and the plurality of polypeptides prior toattempting to determine or to detect the presence of a selectiveaffinity interaction. Such a wash step may be particularly desirablewhen the IL17RLP polypeptides or IL17RLP-like polypeptides or theplurality of polypeptides is bound to a solid support.

The plurality of molecules provided according to this method may beprovided by way of diversity libraries, such as random or combinatorialpeptide or nonpeptide libraries which can be screened for molecules thatspecifically bind IL17RLP polypeptides. Many libraries are known in theart that can be used, e.g., chemically synthesized libraries,recombinant (e.g., phage display libraries), and in vitrotranslation-based libraries. Examples of chemically synthesizedlibraries are described in Fodor et al., 1991, Science 251:767-773;Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature354:82-84; Medynski, 1994, Bio/Technology 12:709-710; Gallop et al.,1994, J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., 1993,Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl.Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques13:412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner,1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.

Examples of phage display libraries are described in Scott and Smith,1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406;Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra,1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65;and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.

In vitro translation-based libraries include but are not limited tothose described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991;and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.

By way of examples of nonpeptide libraries, a benzodiazepine library(see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712)can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc.Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example ofa library that can be used, in which the amide functionalities inpeptides have been permethylated to generate a chemically transformedcombinatorial library, is described by Ostresh et al. (1994, Proc. Natl.Acad. Sci. USA 91:11138-11142).

The variety of non-peptide libraries that are useful in the presentinvention is great For example, Ecker and Crooke, 1995, Bio/Technology13:351-360 list benzodiazepines, hydantoins, piperazinediones,biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids,acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, andoxazolones as among the chemical species that form the basis of variouslibraries.

Non-peptide libraries can be classified broadly into two types:decorated monomers and oligomers. Decorated monomer libraries employ arelatively simple scaffold structure upon which a variety functionalgroups is added. Often the scaffold will be a molecule with a knownuseful pharmacological activity. For example, the scaffold might be thebenzodiazepine structure.

Non-peptide oligomer libraries utilize a large number of monomers thatare assembled together in ways that create new shapes that depend on theorder of the monomers. Among the monomer units that have been used arecarbamates, pyrrolinones, and morpholinos. Peptoids, peptide-likeoligomers in which the side chain is attached to the alpha amino grouprather than the alpha carbon, form the basis of another version ofnon-peptide oligomer libraries. The first non-peptide oligomer librariesutilized a single type of monomer and thus contained a repeatingbackbone. Recent libraries have utilized more than one monomer, givingthe libraries added flexibility.

Screening the libraries can be accomplished by any of a variety ofcommonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley and Smith, 1989, Adv.Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390;Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992,Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992,Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No.5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all toLadner et al.; Rebar and Pabo, 1993, Science 263:671-673; and CTPublication No. WO 94/18318.

In a specific embodiment, screening to identify a molecule that bindsIL17RLP polypeptides can be carried out by contacting the librarymembers with a IL17RLP polypeptides or IL17RLP-like polypeptidesimmobilized on a solid phase and harvesting those library members thatbind to the IL17RLP polypeptides or IL17RLP-like polypeptides. Examplesof such screening methods, termed “panning” techniques are described byway of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes etal., 1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318;and in references cited herein.

In another embodiment, the two-hybrid system for selecting interactingproteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien etal., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used toidentify molecules that specifically bind to IL17RLP polypeptides orIL17RLP-like polypeptides.

Where the IL17RLP binding molecule is a polypeptide, the polypeptide canbe conveniently selected from any peptide library, including randompeptide libraries, combinatorial peptide libraries, or biased peptidelibraries. The term “biased” is used herein to mean that the method ofgenerating the library is manipulated so as to restrict one or moreparameters that govern the diversity of the resulting collection ofmolecules, in this case peptides.

Thus, a truly random peptide library would generate a collection ofpeptides in which the probability of finding a particular amino acid ata given position of the peptide is the same for all 20 amino acids. Abias can be introduced into the library, however, by specifying, forexample, that a lysine occur every fifth amino acid or that positions 4,8, and 9 of a decapeptide library be fixed to include only arginine.Clearly, many types of biases can be contemplated, and the presentinvention is not restricted to any particular bias. Furthermore, thepresent invention contemplates specific types of peptide libraries, suchas phage displayed peptide libraries and those that utilize a DNAconstruct comprising a lambda phage vector with a DNA insert.

As mentioned above, in the case of a IL17RLP binding molecule that is apolypeptide, the polypeptide may have about 6 to less than about 60amino acid residues, preferably about 6 to about 10 amino acid residues,and most preferably, about 6 to about 22 amino acids. In anotherembodiment, a IL17RLP binding polypeptide has in the range of 15-100amino acids, or 20-50 amino acids.

The selected IL17RLP binding polypeptide can be obtained by chemicalsynthesis or recombinant expression.

EXAMPLES

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting. Many of thefollowing examples are set forth referring specifically to IL17RLPpolynucleotides and polypeptides of the invention. Each example may alsobe practiced to generate and/or examine IL17RLP polynucleotides and/orpolypeptides of the invention.

Example 1(a) Expression and Purification of “His-Tagged” IL17RLP in E.coli

The bacterial expression vector pQE9 (pD10) is used for bacterialexpression in this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311). pQE9 encodes ampicillin antibiotic resistance (“Ampr”)and contains a bacterial origin of replication (“ori”), an WIG induciblepromoter, a ribosome binding site (“RBS”), six codons encoding histidineresidues that allow affinity purification usingnickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN,Inc., supra, and suitable single restriction enzyme cleavage sites.These elements are arranged such that an inserted DNA fragment encodinga polypeptide expresses that polypeptide with the six His residues(i.e., a “6×His tag”) covalently linked to the amino terminus of thatpolypeptide.

The DNA sequence encoding the desired portion of the IL17RLP proteincomprising the extracellular domain of the IL17RLP amino acid sequenceis amplified from the deposited cDNA clone using PCR oligonucleotideprimers which anneal to the amino terminal sequences of the desiredportion of the IL17RLP protein and to sequences in the depositedconstruct 3′ to the cDNA coding sequence. Additional nucleotidescontaining restriction sites to facilitate cloning in the pQE9 vectorare added to the 5′ and 3′ primer sequences, respectively.

For cloning the extracellular domain of the IL17RLP protein, the 5′primer has the sequence 5′ CGC CCA TGG CCG ACC GTT CAA TGT GGC TCT GAAAC 3′ (SEQ ID NO:6) containing the underlined Nco I restriction sitefollowed by 26 nucleotides of the amino terminal coding sequence of themature IL17RLP sequence in SEQ ID NO:2. One of ordinary skill in the artwould appreciate, of course, that the point in the protein codingsequence where the 5′ primer begins may be varied to amplify a DNAsegment encoding any desired portion of the complete IL17RLP proteinshorter or longer than the extracellular domain of the protein. The 3′primer has the sequence 5′ CGC AAG CTT CCA GCC TCC CGG CTT GC 3′ (SEQ IDNO:7) containing the underlined Hind III restriction site followed by 17nucleotides complementary to the 3′ end of the coding sequence of theIL17RLP DNA sequence in FIGS. 1A, 1B, and 1C.

The amplified IL17RLP DNA fragment and the vector pQE9 are digested withNco I and Hind III and the digested DNAs are then ligated together.Insertion of the IL17RLP DNA into the restricted pQE9 vector places theIL17RLP protein coding region downstream from the IPTG-induciblepromoter and in-frame with an initiating AUG and the six histidinecodons.

The ligation mixture is transformed into competent E coif cells usingstandard procedures such as those described by Sambrook and colleagues(Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance (“Kanr”),is used in carrying out the illustrative example described herein. Thisstrain, which is only one of many that are suitable for expressingIL17RLP protein, is available commercially (QIAGEN, Inc., supra).Transformants are identified by their ability to grow on LB plates inthe presence of ampicillin and kanamycin. Plasmid DNA is isolated fromresistant colonies and the identity of the cloned DNA confirmed byrestriction analysis, PCR and DNA sequencing.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml). The O/N culture is used to inoculate a largeculture, at a dilution of approximately 1:25 to 1:250. The cells aregrown to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-β-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lad repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

The cells are then stirred for 3-4 hours at 4° C. in 6M guanidine-HCl,pH 8. The cell debris is removed by centrifugation, and the supernatantcontaining the IL17RLP is loaded onto a nickel-nitrilo-tri-acetic acid(“Ni-NTA”) affinity resin column (QIAGEN, Inc., supra). Proteins with a6×His tag bind to the Ni-NTA resin with high affinity and can bepurified in a simple one-step procedure (for details see: TheQIAexpressionist, 1995, QIAGEN, Inc., supra). Briefly the supernatant isloaded onto the column in 6 M guanidine-HCl, pH 8, the column is firstwashed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10volumes of 6 M guanidine-HCl pH 6, and finally the IL17RLP is elutedwith 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins can be eluted by the addition of 250 mMimmidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4° C. or frozen at −80° C.

The following alternative method may be used to purify IL17RLP expressedin E. coli when it is present in the form of inclusion bodies. Unlessotherwise specified, all of the following steps are conducted at 4-10°C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10° C. and the cells are harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells ware then lysed by passing the solution through amicrofluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the IL17RLPpolypeptide-containing supernatant is incubated at 4° C. overnight toallow further GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4° C. without mixing for 12 hours prior tofurther purification steps.

To clarify the refolded IL17RLP polypeptide solution, a previouslyprepared tangential filtration unit equipped with 0.16 μm membranefilter with appropriate surface area (e.g., Filtron), equilibrated with40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loadedonto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems).The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in astepwise manner. The absorbance at 280 mm of the effluent iscontinuously monitored. Fractions are collected and further analyzed bySDS-PAGE.

Fractions containing the IL17RLP polypeptide are then pooled and mixedwith 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perseptive Biosystems) and weak anion (Poros CM-20, PerseptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A₂₈₀ monitoring of the effluent. Fractionscontaining the IL17RLP polypeptide (determined, for instance, by 16%SDS-PAGE) are then pooled.

The resultant IL17RLP polypeptide exhibits greater than 95% purity afterthe above refolding and purification steps. No major contaminant bandsare observed from Commassie blue stained 16% SDS-PAGE gel when 5 μg ofpurified protein is loaded. The purified protein is also tested forendotoxin/LPS contamination, and typically the LPS content is less than0.1 ng/ml according to LAL assays.

Example 2 Cloning and Expression of IL17RLP Protein in a BaculovirusExpression System

In this illustrative example, the plasmid shuttle vector pA2 is used toinsert the cloned DNA encoding complete protein, including its naturallyassociated secretory signal (leader) sequence, into a baculovirus toexpress the mature IL17RLP protein, using standard methods as describedby Summers and colleagues (A Manual of Methods for Baculovirus Vectorsand Insect Cell Culture Procedures, Texas Agricultural ExperimentalStation Bulletin No. 1555 (1987)). This expression vector contains thestrong polyhedrin promoter of the Autographa californica nuclearpolyhedrosis virus (AcMNPV) followed by convenient restriction sitessuch as Bam HI, Xba I and Asp 718. The polyadenylation site of thesimian virus 40 (“SV40”) is used for efficient polyadenylation. For easyselection of recombinant virus, the plasmid contains thebeta-galactosidase gene from E. coli under control of a weak Drosophilapromoter in the same orientation, followed by the polyadenylation signalof the polyhedrin gene. The inserted genes are flanked on both sides byviral sequences for cell-mediated homologous recombination withwild-type viral DNA to generate a viable virus that express the clonedpolynucleotide.

Many other baculovirus vectors could be used in place of the vectorabove, such as pAc373, pVL941 and pAcIM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, by Luckow andcoworkers (Virology 170:31-39 (1989)).

The cDNA sequence encoding the extracellular domain of the IL17RLPprotein in the deposited clone, including the AUG initiation codon andthe naturally associated leader sequence shown in SEQ ID NO:2, isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ sequences of the gene. The 5′ primer has the sequence 5′ CGC GGA TCCATG TCG CTC GTG CTG CTA AGC CTG G 3′ (SEQ ID NO:8) containing theunderlined Bam HI restriction enzyme site, an efficient signal forinitiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol.196:947-950 (1987)), followed by 25 of nucleotides of the sequence ofthe complete IL17RLP protein shown in FIGS. 1A, 1B, and 1C, beginningwith the AUG initiation codon. The 3′ primer has the sequence 5′ CGC GGTACC CCA GCC TCC CGG CTT GC 3′ (SEQ ID NO:9) containing the underlinedAsp 718 restriction site followed by 17 nucleotides complementary to the3′ noncoding sequence in FIGS. 1A, 1B, and 1C.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“GENECLEAN™,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with Bam HI and Asp 718 and againis purified on a 1% agarose gel. This fragment is designated herein F1.

The plasmid is digested with the restriction enzymes Bam HI and Asp 718and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“GENECLEAN™” BIO 101 Inc., La Jolla, Calif.). This vector DNA isdesignated herein “V1”.

Fragment F1 and the dephosphorylated plasmid V1 are ligated togetherwith T4 DNA ligase. E. coli H13101 or other suitable E. coli hosts suchas XL-1 Blue (Statagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria are identified that contain the plasmid with the human IL17RLPgene by digesting DNA from individual colonies using Bam HI and Asp 718and then analyzing the digestion product by gel electrophoresis. Thesequence of the cloned fragment is confirmed by DNA sequencing. Thisplasmid is designated herein pA2IL17RLP.

Five μg of the plasmid pA2IL17RLP is co-transfected with 1.0 μg of acommercially available linearized baculovirus DNA (“BACULOGOLD™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Feigner and colleaguew (Proc. Natl. Acad. Sci. USA84:7413-7417 (1987)). One μg of BACULOGOLD™ virus DNA and 5 μg of theplasmid pA2IL17RLP are mixed in a sterile well of a microtiter platecontaining 50 μl of serum-free Grace's medium (LIFE TECHNOLOGIES™ Inc.,Gaithersburg, Md.). Afterwards, 1.0 μl LIPOFECTIN™ plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC™ CRL 1711) seeded in a 35 mm tissue culture platewith 1 ml Grace's medium without serum. The plate is then incubated for5 hours at 27° C. The transfection solution is then removed from theplate and 1 ml of Grace's insect medium supplemented with 10% fetal calfserum is added. Cultivation is then continued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith (supra). An agarose gelwith “Blue Gal” (LIFE TECHNOLOGIES™ Inc., Gaithersburg) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by LIFE TECHNOLOGIES™ Inc.,Gaithersburg, page 9-10). After appropriate incubation, blue stainedplaques are picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C. Therecombinant virus is called V-IL17RLP.

To verify the expression of the IL17RLP gene Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus V-IL17RLP at a multiplicity ofinfection (“MOI”) of about 2. If radiolabeled proteins are desired, 6hours later the medium is removed and is replaced with SF900 II mediumminus methionine and cysteine (available from LIFE TECHNOLOGIES™ Inc.,Rockville, Md.). After 42 hours, 5 μCi of ³⁵S-methionine and 5 μCi³⁵S-cysteine (available from Amersham) are added. The cells are furtherincubated for 16 hours and then are harvested by centrifugation. Theproteins in the supernatant as well as the intracellular proteins areanalyzed by SDS-PAGE followed by autoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe extracellular domain of the IL17RLP protein, and thus the cleavagepoint and length of the naturally associated secretory signal peptide.

Example 3 Cloning and Expression of IL17RLP in Mammalian Cells

A typical mammalian expression vector contains the promoter element,which mediates the initiation of transcription of mRNA, the proteincoding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as pSVL and pMSG(PHARMACIA™, Uppsala, Sweden), pRSVcat (ATCC™ 37152), pSV2dhfr (ATCC™37146) and pBC12M1 (ATCC™ 67109). Mammalian host cells that could beused include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 andC127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, hygromycin allows theidentification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulto develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS; Murphy, et al., Biochem J.227:277-279 (1991); Bebbington, et al., Bio/Technology 10:169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of proteins.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen, et al., Mol. Cell. Biol. 5:438-447(1985)) plus a fragment of the CMV-enhancer (Boshart, et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites Bam HI, Xba I and Asp 718, facilitate the cloningof the gene of interest. The vectors contain in addition the 3′ intron,the polyadenylation and termination signal of the rat preproinsulingene.

Example 3(a) Cloning and Expression in COS Cells

The expression plasmid, pIL17RLPHA, is made by cloning a portion of thecDNA encoding the extracelluar domain of the IL17RLP protein into theexpression vector pcDNAI/Amp or pcDNAIII (which can be obtained fromInvitrogen, Inc.).

The expression vector pcDNAI/amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a cDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson and colleagues (Cell 37:767 (1984)). The fusion of the HA tag tothe target protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

A DNA fragment encoding the extracellular domain of the IL17RLPpolypeptide is cloned into the polylinker region of the vector so thatrecombinant protein expression is directed by the CMV promoter. Theplasmid construction strategy is as follows. The IL17RLP cDNA of thedeposited clone is amplified using primers that contain convenientrestriction sites, much as described above for construction of vectorsfor expression of IL17RLP in E. coli. Suitable primers include thefollowing, which are used in this example. The 5′ primer, containing theunderlined Bam HI site, a Kozak sequence, an AUG start codon, and 25nucleotides of the 5′ coding region of the extracellular domain of theIL17RLP polypeptide, has the following sequence: 5′ GCC GGA TCC GCC ACCATG AAC TCC TTC TCC ACA AGC GCC TTC GGT CCA GTT GCC TTC TCC CTG GGG CTGCTC CTG GTG TTG CCT GCT GCC TTC CCT GCC CCA GTA TGT CGC TCG TGC TGC TAAGCC TGG 3′ (SEQ ID NO: 10). The 3′ primer, containing the underlined Asp718 and 17 of nucleotides complementary to the 3′ coding sequenceimmediately before the stop codon, has the following sequence: 5′ GGCCGG GTA CCC CAG CCT CCC GGC TTG C 3′ (SEQ ID NO:11).

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith Bam HI and Asp 718 and then ligated. The ligation mixture istransformed into E. coli strain SURE (STRATAGENE™ Cloning Systems, LaJolla, Calif. 92037), and the transformed culture is plated onampicillin media plates which then are incubated to allow growth ofampicillin resistant colonies. Plasmid DNA is isolated from resistantcolonies and examined by restriction analysis or other means for thepresence of the fragment encoding the extracellular domain of theIL17RLP polypeptide

For expression of recombinant IL17RLP, COS cells are transfected with anexpression vector, as described above, using DEAE-dextran, as described,for instance, by Sambrook and coworkers (Molecular Cloning: a LaboratoryManual, Cold Spring Laboratory Press, Cold Spring Harbor, N.Y. (1989)).Cells are incubated under conditions for expression of IL17RLP by thevector.

Expression of the IL17RLP-HA fusion protein is detected by radiolabelingand immunoprecipitation, using methods described in, for example Harlowand colleagues (Antibodies: A Laboratory Manual, 2nd Ed.; Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)). To this end,two days after transfection, the cells are labeled by incubation inmedia containing ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and the lysed withdetergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1%NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson andcolleagues (supra). Proteins are precipitated from the cell lysate andfrom the culture media using an HA-specific monoclonal antibody. Theprecipitated proteins then are analyzed by SDS-PAGE and autoradiography.An expression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

Example 3(b) Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of IL17RLP polypeptide.Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC™ AccessionNo. 37146). The plasmid contains the mouse DHFR gene under control ofthe SV40 early promoter. Chinese hamster ovary- or other cells lackingdihydrofolate activity that are transfected with these plasmids can beselected by growing the cells in a selective medium (alpha minus MEM,LIFE TECHNOLOGIES™) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., Alt, F. W., etal., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C.Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. andSydenham, M. A. Biotechnology 9:64-68 (1991)). Cells grown in increasingconcentrations of MTX develop resistance to the drug by overproducingthe target enzyme, DHFR, as a result of amplification of the DHFR gene.If a second gene is linked to the DHFR gene, it is usually co-amplifiedand over-expressed. It is known in the art that this approach may beused to develop cell lines carrying more than 1,000 copies of theamplified gene(s). Subsequently, when the methotrexate is withdrawn,cell lines are obtained which contain the amplified gene integrated intoone or more chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus(Cullen, et al., Mol. Cell. Biol. 5:438-447 (1985)) plus a fragmentisolated from the enhancer of the immediate early gene of humancytomegalovirus (CMV; Boshart, et al., Cell 41:521-530 (1985)).Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: Bam HI, Xba I,and Asp 718. Behind these cloning sites the plasmid contains the 3′intron and polyadenylation site of the rat preproinsulin gene. Otherhigh efficiency promoters can also be used for the expression, e.g., thehuman beta-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.CLONTECH™'s Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the IL17RLP polypeptide in a regulatedway in mammalian cells (Gossen, M., and Bujard, H. Proc. Natl. Acad.Sci. USA 89:5547-5551 (1992)). For the polyadenylation of the mRNA othersignals, e.g., from the human growth hormone or globin genes can be usedas well. Stable cell lines carrying a gene of interest integrated intothe chromosomes can also be selected upon co-transfection with aselectable marker such as gpt, G418 or hygromycin. It is advantageous touse more than one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC4 is digested with the restriction enzymes Bam HI and Asp718 and then dephosphorylated using calf intestinal phosphates byprocedures known in the art. The vector is then isolated from a 1%agarose gel.

The DNA sequence encoding the extracellular domain of the IL17RLPpolypeptide is amplified using PCR oligonucleotide primers correspondingto the 5′ and 3′ sequences of the desired portion of the gene. The 5′primer containing the underlined Bam HI site, a Kozak sequence, an AUGstart codon, and 25 nucleotides of the 5′ coding region of theextracellular domain of the IL17RLP polypeptide, has the followingsequence: 5′ CTA GCC GGA TCC GCC ACC ATG TCG CTC GTG CTG CTA AGC CTG G3′ (SEQ ID NO:12). The 3′ primer, containing the underlined Asp 718 and17 of nucleotides complementary to the 3′ coding sequence immediatelybefore the stop codon as shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1),has the following sequence: 5′ GGC CGG GTA CCC CAG CCT CCC GGC TTG C 3′(SEQ ID NO:13).

The amplified fragment is digested with the endonucleases Bam HI and Asp718 and then purified again on a 1% agarose gel. The isolated fragmentand the dephosphorylated vector are then ligated with T4 DNA ligase. E.coli HB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. Five μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSVneo using LIPOFECTIN™ (Feigner, et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 4 Tissue Distribution of IL17RLP mRNA Expression

Northern blot analysis is carried out to examine IL17RLP gene expressionin human tissues, using methods described by, among others, Sambrook andcolleagues (supra). A cDNA probe containing the entire nucleotidesequence of the IL17RLP protein (SEQ ID NO:1) is labeled with ³²P usingthe REDIPRIME™ DNA labeling system (Amersham Life Science), according tomanufacturer's instructions. After labeling, the probe is purified usinga CHROMA SPIN-100™ column (CLONTECH™ Laboratories, Inc.), according tomanufacturer's protocol number PTI200-L The purified labeled probe isthen used to examine various human tissues for IL17RLP mRNA.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) are obtained from CLONTECH™ andare examined with the labeled probe using EXPRESSHYB™ hybridizationsolution (CLONTECH™) according to manufacturer's protocol numberPT1190-1. Following hybridization and washing, the blots are mounted andexposed to film at −70° C. overnight, and films developed according tostandard procedures.

In Northern blot experiments performed essentially as described above,expression of the IL17RLP transcript was detected in pancreas, kidney,liver, and fetal liver. Lower expression was also observed in otherendocrine organs such as testis, colon, and small intestine. See also,Example 12.

Example 5 Blocking Effect of Soluble IL17RLP on IL-20-Induced NeutrophilMigration and Macrophage Activation in the Mouse Peritoneum

An analysis of the use of soluble IL17RLP (“sIL17RLP”) as ananti-inflammatory agent is performed through the use of a human IL-20(“hIL-20”)-induced inflammation model in mice. Our recent experimentindicate that, when given intraperitoneally, hIL-20 induces asignificant migration of neutrophils into the mouse peritoneum at 4hours after injection as observed by both FACS and Wright-Giemsa stainedcytospin analysis. In addition, after hIL-20 challenge, peritonealmacrophages show activation signals by morphology. Soluble sIL17RLP isexpected to bind hIL-20 and inhibit hIL-20-induced neutrophil migrationand macrophage activation.

Initiation of the inflammation condition is induced by a singleintraperitoneal injection of high (25 μg) and low doses (1-10 μg) ofhIL-20 into BALB/c mice. Groups of 4 mice receive either 0.1 to 10 mg/kgof sIL17RLP, soluble human IL-17 receptor or negative control humanreceptor, intraperitoneally once between 0 and 2 hours prior to hIL-20injection. The effect of sIL17RLP on neutrophil migration and macrophageactivation in the peritoneum is analyzed at 4, 16, 24 or 48 hours byFACS and cytospin method. Briefly, for FACS analysis, collectedperitoneal cells are stained with fluorescein phycoerythrin-conjugatedantibodies against MHC class II (I-A/I-E) and FITC-conjugated anti-Mac-1or anti-Gr1 (PharMingen (San Diego, Calif.)). Cells are then analyzed ona FACScan (Becton Dickinson, San Jose, Calif.), and the percentages ofI-A/I-E hi+Mac-1+ macrophages and Gr1+ neutrophils are determined bytwo-color analysis. For cytospin method, peritoneal cells are spun downon to microscope slides and then differentiated by Wright-Giemsastaining. The percentages of activated macrophages and neutrophils aredetermined according to the cell morphology.

Example 6 Effect of Soluble IL17RLP on Adjuvant-Induced Arthritis

An analysis of the use of soluble IL17RLP (“sIL17RLP”) to treatrheumatoid arthritis (RA) is performed through the use of anadjuvant-induced arthritis model (AIA) in rats. AIA is awell-characterized and reproducible animal model of rheumatoidarthritis, which is well known to one of ordinary skill in the art(Pearson, et al. Ann. Rheum. Dis. 15:379, (1956)); Pearson, et al.,Arthritis Rheum. 2:440, (1959)). sIL17RLP is expected to bind to hIL-20and inhibit IL-20-induced synoviocyte activation and cytokineproduction, which may involve in the perpetuation of chronic arthritis.Lewis rats (available from Charles River Lab, Raleigh, N.C.) are used asthe common and responsive strains for adjuvant-induced arthritis inthese experiments.

Initiation of the arthritis condition is induced by the intradermalinjection of 0.1 ml adjuvant (5 mg/ml) into the base of the tail. Groupsof 5 to 6 rats received either 0.1 to 10 mg/kg sIL17RLP or vehicleintra-articularly 10 days after the injection of adjuvant when the acuteinflammation just begins. The effect of sIL17RLP on chronic arthritis isanalyzed radiologically once each week between day 15-30 essentially asdescribed by Taurog and colleagues (J. Exp. Med. 162:962, (1985)).Briefly, rats are anesthetized with ether or chloral hydrate andpositioned so that both hind limbs are X-rayed together. The X-ray filmsare examined blindly using a scoring system of 0-3 for periostealreaction, bony erosions, joint space narrowing and destruction. Whenthere is a significant amount of joint damage in vehicle-treated rats,the animals are sacrificed. At this point, the paws are evaluatedhistologically for the relative degree of tissue damage and for thetherapeutic effect sIL17RLP has elicited on these joints. Finally,sIL17RLP- and vehicle-treated animals undergo a clinical evaluationtwice per week to assess hind paw volume using a plethysmometer systemand body weight.

Alternatively, rheumatoid synoviocytes are isolated from RA patientsundergoing knee or wrist synovectomy and cultured in 150 cm² flasks.Nonadherent cells are removed and adherent cells are trypsinized atconfluence and passaged. Synoviocytes used between passages 3 and 8constitute a homogenous population of fibroblast-like cells.Synoviocytes are cultured in 96-well plates in a final volume of 200 μlof the medium. Human IL-20 polypeptides (or human IL-17 as a control)are added at different concentrations to the medium at the onset of theculture. In experimental flasks, human sIL17RLP polypeptide is alsoadded to the culture medium. Subsequently, cell-free supernatants arecollected after 72 hr, and stored at −20° C. for further use in cytokineassays. Concentrations of IL-6 and IL-8 are measured by ELISA. Adecrease in IL-6 and/or IL-8 levels in the culture supernatant indicatesthat the sIL17RLP polypeptide inhibits the IL-20-mediated increase inIL-6 and/or IL-8 production in this culture system. Consequently,sIL17RLP may be useful to treat rheumatoid arthritis and other relatedimmunoregulatory disorders and diseases.

Example 7 Effect of Soluble IL17RLP in Treating Graft Versus HostDisease in Mice

An analysis of the use of soluble IL17RLP (“sIL17RLP”) to treatgraft-versus-host disease (GVHD) is performed through the use of aC57BL/6 parent into (BALB/c×C57BL/6) F1 mouse model. This parent into F1mouse model is a well-characterized and reproducible animal model ofGVHD in bone marrow transplant patients, which is well know to one ofordinary skill in the art (see, Gleichemann, et al., Immunol. Today5:324, (1984)). IL17RLP is structurally related to the IL-17R which, insoluble form, has a beneficial effect on the prolongation of allograftsurvival in association with its inhibiting effect onalloantigen-induced lymphocyte proliferation. sIL17RLP is expected toinhibit the activation of the donor T cells to host MHC class II antigen(alloantigen) which play a crucial role in the pathogenesis of GVHD.

Initiation of the experimental GVHD condition is induced by theintravenous injection of ˜1-3×10⁸ spleen cells from C57BL/6 mice into(BALB/c×C57BL/6) F1 mice (available from Jackson Lab, Bar Harbor, Me.).Groups of 6 to 8 mice received either 0.1 to 5.0 mg/kg of sIL17RLP ornegative control intraperitoneally daily following the injection ofspleen cells. The effect of sIL17RLP on lymphoid hypoplasia and atrophyof spleen is analyzed by FACS and histopathology at multiple time points(3-4) between days 10 and 30. Briefly, splenocytes are prepared fromnormal CBF1 mice, GVHD mice or sIL17RLP-treated mice, and stained withfluorescein phycoerythrin-conjugated anti-H-2 Kb, biotin-conjugatedanti-14-2 Kd, and FITC-conjugated anti-CD4, anti-CD8, or anti-B220,followed by a CyChrome-conjugated avidin (PharMingen (San Diego,Calif.)). Cells are then analysis on a FACScan (Becton Dickinson, SanJose, Calif.). Recipient and donor lymphocytes are identified as H-2Kb+Kd+ and H-1-2 Kb+ Kd− cells, respectively. Cell numbers of CD4+T,CD8+ T and B220+ B cells of recipient or donor origin are calculatedfrom the total numbers of splenocytes recovered and the percentages ofeach subpopulation are determined by the three color analysis.Histological evaluation of the relative degree of tissue damage in otherGVHD-associated organs (liver, skin and intestine) may be conductedafter sacrificing the animals for the beneficial potential of sIL17RLPon these organs.

In addition, the effect of sIL17RLP on spontaneous proliferation andIL-2 production of host splenocytes is analyzed between day 2-10.Finally, sIL17RLP— and its negative control-treated animals undergo aclinical evaluation every other day to assess cachexia, body weight andlethality. Soluble sIL17RLP in combination therapy withimmunosuppressive agents may also be examined in this GVHD murine model.

Example 8 Analysis of IL-17RLP Ligand Candidates

IL17RLP ligand candidates are screened for binding using BIACORE™technology which enables one to monitor binding events between two ormore molecules, in real time, without the use of labels. BIACORE™technology relies on the phenomenon of surface plasmon resonance (SPR)which occurs when surface plasmon waves are excited at a metal/liquidinterface. Light is directed at, and reflected from, the side of thesurface not in contact with sample, and SPR causes a reduction in thereflected light intensity at a specific combination of angle andwavelength. Biomolecular binding events cause changes in the refractiveindex at the surface layer, which are detected as changes in the SPRsignal.

The conditioned culture supernatants from three IL-20 CHO (see copendingU.S. patent application Ser. No. 09/115,832) clones (numbers 10, 16 and22), as well as, IL-17 (purchased from R&D) were analyzed for binding toIL17-like receptors. The data indicate that compared to the negativecontrol conditioned media (pC4 vector alone) that all clones showedgreater binding. The binding was approximately 115 RU for clones 16 and22, ˜65 RU for clone 10 and ˜20 RU for pC4. This binding was greaterthan that found for IL-17 which was ˜60 RU measured at 25 ug/mL. Theexact concentration of IL-20 in the culture supernatants is not knownbut is estimated to be comparable to IL-17, i.e., ˜25 ug/mL. This resultsuggests that the IL-17 receptor binds both ligands, and may even bindIL-20 better.

The binding of IL-20 and IL-17 to IL-17 receptor (IL 17R-Fc) and IL17RLPfused to the human immunoglobulin domain (IL17RLP-Fc) afterimmobilization of the receptor on a BIACORE™ flow cell. Two CHO cellIL-20 preparations were first analyzed as they contain differentN-terminal forms of the protein. IL-17 (R&D) ligand was also analyzed.The results indicate that IL-20 predominately bound to IL17RLP-Fc and toa much lesser extent to IL-17R. The dissociation of IL-20 from theIL17RLP-Fc appeared to be biphasic for both batches which might be dueto the presence different N-terminally truncated forms of the proteinpresent in both batches. In contrast, IL-17 bound almost exclusively tothe IL-17R which little or no binding to IL17RLP-Fc.

Thus, these results suggest that IL-20 interacts with the IL-17 receptorand the IL17RLP described herein. As a result, IL17RLP, or solublefragments thereof, may be useful to modulate the receptor activationpathways in which these receptors are involved. IL17RLP polypeptides ofthe invention may be used as an antagonist for binding IL-20polypeptides and/or other related or unrelated polypeptides whichinteract with this receptor or the IL-20 ligand, e.g., IL-17. IL17RLPpolypeptides of the invention may thus be useful in the diagnosis and/ortreatment of immune disorders involving the IL-17 and IL17RLP moleculesas known in the art and as described above.

Example 9 Gene Therapy Using the Endogenous IL17RLP Gene

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous IL17RLP sequence with apromoter via homologous recombination as described, for example, in U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International PublicationNumber WO 96/29411, published Sep. 26, 1996; International PublicationNumber WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl.Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature342:435-438 (1989). This method involves the activation of a gene whichis present in the target cells, but which is not expressed in the cells,or is expressed at a lower level than desired. Polynucleotide constructsare made which contain a promoter and targeting sequences, which arehomologous to the 5′ non-coding sequence of endogenous IL17RLP, flankingthe promoter. The targeting sequence will be sufficiently near the 5′end of IL17RLP so the promoter will be operably linked to the endogenoussequence upon homologous recombination. The promoter and the targetingsequences can be amplified using PCR. Preferably, the amplified promotercontains distinct restriction enzyme sites on the 5′ and 3′ ends.Preferably, the 3′ end of the first targeting sequence contains the samerestriction enzyme site as the 5′ end of the amplified promoter and the5′ end of the second targeting sequence contains the same restrictionsite as the 3′ end of the amplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenousIL17RLP sequence. This results in the expression of IL17RLP in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM+10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEMS pH 7.3, 137 mMNaCl, 5 mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the IL17RLP locus, plasmid pUC18(MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMVpromoter is amplified by PCR with an XbaI site on the 5′ end and a BamHIsite on the 3′ end. Two IL17RLP non-coding sequences are amplified viaPCR: one IL17RLP non-coding sequence (IL17RLP fragment 1) is amplifiedwith a HindIII site at the 5′ end and an Xba site at the 3′ end; theother IL17RLP non-coding sequence (IL17RLP fragment 2) is amplified witha BamHI site at the 5′ end and a HindIII site at the 3′ end. The CMVpromoter and IL17RLP fragments are digested with the appropriate enzymes(CMV promoter—XbaI and BamHI; IL17RLP fragment 1-XbaI; IL17RLP fragment2-BamHI) and ligated together. The resulting ligation product isdigested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1.5×10⁶ cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and250-300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37° C.

The following day, the media is aspirated and replaced with 10 ml offresh media and incubated for a further 16-24 hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

Example 10 Production of an Antibody

Hybridoma Technology

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing IL17RLP are administered to an animal toinduce the production of sera containing polyclonal antibodies. In apreferred method, a preparation of IL17RLP protein is prepared andpurified to render it substantially free of natural contaminants. Such apreparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

Monoclonal antibodies specific for protein IL17RLP are prepared usinghybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler etal., Eur. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292(1976); Hammerling et al in: Monoclonal Antibodies and T-CellHybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal(preferably a mouse) is immunized with IL17RLP polypeptide or, morepreferably, with a secreted IL17RLP polypeptide-expressing cell. Suchpolypeptide-expressing cells are cultured in any suitable tissue culturemedium, preferably in Earle's modified Eagle's medium supplemented with10% fetal bovine serum (inactivated at about 56° C.), and supplementedwith about 10 g/l of nonessential amino acids, about 1,000 U/ml ofpenicillin, and about 100 μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP2O), available from the ATCC™.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981). The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the IL17RLP polypeptide.

Alternatively, additional antibodies capable of binding to IL17RLPpolypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodywhich binds to a second antibody. In accordance with this method,protein specific antibodies are used to immunize an animal, preferably amouse. The splenocytes of such an animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify cloneswhich produce an antibody whose ability to bind to the IL17RLPprotein-specific antibody can be blocked by IL17RLP. Such antibodiescomprise anti-idiotypic antibodies to the IL17RLP protein-specificantibody and are used to immunize an animal to induce formation offurther IL17RLP protein-specific antibodies.

For in vivo use of antibodies in humans, an antibody is “humanized”.Such antibodies can be produced using genetic constructs derived fromhybridoma cells producing the monoclonal antibodies described above.Methods for producing chimeric and humanized antibodies are known in theart and are discussed infra. (See, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature314:268 (1985).)

Isolation of Antibody Fragments Directed Against IL17RLP From a Libraryof scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a library of antibody fragments which contain reactivities againstIL17RLP to which the donor may or may not have been exposed (see e.g.,U.S. Pat. No. 5,885,793 incorporated herein by reference in itsentirety).

Rescue of the Library. A library of scFvs is constructed from the RNA ofhuman PBLs as described in PCT publication WO 92/01047. To rescue phagedisplaying antibody fragments, approximately 109 E. coli harboring thephagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 withshaking. Five ml of this culture is used to innoculate 50 ml of2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, seePCT publication WO 92/01047) are added and the culture incubated at 37°C. for 45 minutes without shaking and then at 37° C. for 45 minutes withshaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and thepellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillinand 50 ug/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C.Phage particles are purified and concentrated from the culture medium bytwo PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBSand passed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 1013 transducing units/ml(ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing.

Example 11 Neutralization of IL17RLP/IL17RLP Ligand (e.g., IL20)Interaction with an Anti-IL17RLP Monoclonal Antibody

Monoclonal antibodies are generated against IL17RLP protein according tothe following method. Briefly, mice are given a subcutaneous injection(front part of the dorsum) of 50 micrograms of His-tagged IL17RLPprotein produced by the method of Example 2 in 100 microliters of PBSemulsified in 100 microliters of complete Freunds adjuvant. Threeadditional subcutaneous injections of 25 micrograms of IL17RLP inincomplete Freunds adjuvant are given at 2-week intervals. The animalsare rested for a month before they received the final intraperitonealboost of 25 micrograms of IL17RLP in PBS. Four days later mice aresacrificed and splenocytes taken for fusion.

The process of “Fusion” is accomplished by fusing splenocytes from onespleen were with 2×10E7 P3X63Ag8.653 plasmacytoma cells using PEG 1500(BOEHRINGER™ Mannheim), according to the manufacturer's modifications ofan earlier described method. (See, Gefter, M. L., et al. Somatic CellGenet. 3:231-36 (1977); BOEHRINGER™ Mannheim, PEG 1500 (Cat. No.783641), product description.)

After fusion, the cells are resuspended in 400 ml of HAT mediumsupplemented with 20% FBS and 4% Hybridoma Supplement (BOEHRINGER™Mannheim) and distributed to 96 well plates at a density of 200microliters per well. At day 7 post-fusion, 100 microliters of medium isaspirated and replaced with 100 microliters of fresh medium. At day 14post-fusion, the hybridomas are screened for antibody production.

Hybridoma supernatants are screened by ELISA for binding to IL17RLPprotein immobilized on plates. Plates are coated with IL17RLP byovernight incubation of 100 microliters per well of IL17RLP in PBS at aconcentration of 2 micrograms per ml. Hybridoma supernatants are diluted1:10 with PBS and placed in individual wells of IL17RLP-coated platesand incubated overnight at 4° C. On the following day, the plates arewashed 3 times with PBS containing 0.1% Tween-20 and developed using theanti-mouse IgG ABC system (Vector Laboratories). The color developmentreaction is stopped with the addition of 25 ml/well of 2M E7SO₄. Theplates are then read at 450 nm.

Hybridoma supernatants are checked for Ig isotype using Isostrips.Cloning is done by the method of limiting dilutions on HT medium. About3×10E6 cells in 0.9 ml of HBSS are injected in pristane-primed mice.After 7-9 days, ascitic fluid is collected using a 19 g needle. Allantibodies are purified by protein G affinity chromatography using theActa FPLC system (PHARMACIA™).

All purified monoclonal antibodies are tested for binding to differentforms of IL17RLP (including His-tagged and protein produced from abaculoviral system (see Example 2)) in both Western blot analysis andELISA. Antibodies are also tested for the ability to capture solubleIL17RLP from solution. Antibodies are tested for the ability todifferentially recognize membrane-bound IL17RLP as compared to a solubleIL17RLP (e.g., an extracellular domain of IL17RLP). Antibodies aretested for the ability to neutralize IL17RLP receptor-ligandinteractions.

Example 12 A Novel Cytokine Receptor-Ligand Pair: Identification,Molecular Characterization, and in Vivo Immunomodulatory Activity

Cytokines are secreted regulatory peptides that mediate a wide range ofbiological activities by binding to specific cell surface receptors ontarget cells. Cytokine actions include control of cell proliferation anddifferentiation, regulation of hemopoiesis, immune and inflammatoryresponses (See e.g., Thomson, A. The Cytokine Handbook, 3rd Ed.,Academic Press, New York, N.Y. (1998)). Cytokines are also majororchestrators of host defense processes and, as such, are involved inresponses to exogenous as well as endogenous insults and in repair orrestoration of tissue integrity.

Except for the presence of an N-terminal signal peptide usually requiredfor secretion, the cytokines known thus far are members of many distinctand structurally unrelated families of molecules.

A novel homologue of Interleukin-17 (“IL-17”) has been identified. (See,International Patent Application No. US98/14609; see also, Li, et al,Proc. Natl. Acad. Sci. 97:773-778 (2000)). IL-17 is a cytokine-inducingglycoprotein of 155 amino acids, produced predominantly by activatedCD4+ T cells and double negative (CD4-CD8-) T cells exhibiting indirectproinflammatory and hematopoietic properties (See e.g., Yao, Z., et al.,J. Immunol. 155:5483-5486 (1995); Fossiez, F., et al, J. Exp. Med.183:2593-2603 (1996); Cai, X. Y., et al., Immunol. Lett. 62:51-58(1998); Chabaud, M., et al, J. Immunol. 161:409-414 (1998); Jovanovic,D. V., et al., J. Immunol. 160:3513-3521 (1998)). In vivo, itsexpression has been reported elevated in the rheumatoid synovium, inmultiple sclerosis blood and cerebrospinal fluid and in peripheral bloodmononuclear cells following ischemic stroke (See e.g., Kotake, S., etal., J. Clin. Invest 103:1345-1352 (1999); Chabaud, M., et al.,Arthritis Rheum. 42:963-970 (1999); Aarvak, T., et al., Scand. J.Immunol. 50:1-9 (1999); Matusevicius, D., et al., Mult. Scler. 5:101-104(1999); Kostulas, N., et al., Stroke 30:2174-2179 (1999)). Is alsoproduced by tumor-infiltrating lymphocytes and increases tumorigenicityof human cervical tumors in nude mice (See, Fridman, W. H., et al., Res.Immunol. 149:7-8 (1998); Tarour, E., et al., Cancer Res. 59:3698-3704(1999)). More recently, IL-17 has been implicated in allergic skinimmune responses (See, Albanesi, C., et al., J. Immunol. 162:494-502(1999)), neutrophil recruitment during airway inflammation (See,Antonysamy, M. A., et al., Human Immunol. 55(Suppl):1-15 (1997)),cardiac and renal allograft rejection (See, Laan, M., et al., J.Immunol. 162:2347-2352 (1999); Van Kooten, C., et al, J. Am. Soc.Nephrol. 9:1526-1534 (1998); Antonysamy, M. A., et al., J. Immunol.162:577-584 (1999)) and granulopoiesis (See, Fine, J. S., et al., J.Allergy Clin. Immunol. 99:225 (1997); Schwarzenberger, P., et al., J.Immunol. 161:6383-6389 (1998)). In addition, it has been found toup-regulate nitric oxide production in human osteoarthritic cartilageand inflammatory cytokine production by rheumatoid arthritissynoviocytes (See, Attur, M. G., et al., Arthritis Rheum. 40:1050-1053(1997); Amin, A. R., et al., Curr. Opin. Rheumatol. 10:263-268 (1998)),to stimulate osteoclastogenesis and the expression of several genesassociated with inflammation and cartilage degradation in humanchondrocytes (See, Lotz, M., et al., Arthritis Rheum. 39(Suppl.):559(1996); Van bezooijen, R. L., et al., J. Bone Miner. Res. 14:1513-1521(1999); Tali Shalom-Barak, T., et al., J. Biol. Chem. 173:27467-27473(1998); Martel-Pelletier, J., et al., Arthritis Rheum. 42:2399-2409(1999)), and to induce ICAM-1 expression in human bronchial epithelialcells (See, Kawaguchi, M., et al., Cell 46:659-667 (1986)).

Sequence and expression analysis—Full-length cDNAs for human IL-20 (SEQID NO:34) and IL17RLP (SEQ ID NOs:1 and 18) were identified, sequenced,and submitted to GenBank. The clones were assigned the accession numbers(AF212311) and (AF212365), respectively. DNA sequencing was performedusing ABI 377 automated DNA sequencers and PE Biosystems Big DyeTerminator sequencing chemistries (Foster City, Calif.). Northern blotanalysis of poly-A RNA samples was performed using CLONTECH™ (Palo Alto,Calif.) multiple tissue Northern blots. For analysis of murine IL-20transcripts, total RNA was prepared from rodent organs, separated onagarose gels containing formamide and blotted onto Nylon filtersMembranes were hybridized overnight in Hybrisol solution (Oncor),preheated to 42° C. before use, followed by two subsequent washes in2×SSC/0.1% SDS and 0.2×SSC/0.1% SDS at the same temperature.Double-stranded cDNA probes, used at a minimum specific activity of2×10⁹ cpm/microgram, were generated by restriction digestion,³²P-labelled using the REDEPRIME™ random primer labelling system(Amersham/Arlington Heights, Ill.) and purified with NucTrap ionexchange push columns (STRATAGENE™, La Jolla, Calif.).

Mapping—The genomic position of the IL20 gene was determined with thestandard G3 radiation hybrid panel (Research Genetics, Huntsville,Ala.). The panel DNAs were amplified by PCR using IL-20 gene specificprimers 5′-GGC GGG CAG CAG CTG CAG GCT GAC C-3′ (SEQ ID NO:19) and5′-CTG GGC TGG CCC AGC CCC AGG AAG-3′ (SEQ ID NO:20). The primers usedfor mapping of IL17RLPR were 5′-GAT CCT CCC GGA CTT CAA GAG GC-3′ (SEQID NO:21) and 3′-GGA AAG GCC AGG CAG GCC TGG-3′ (SEQ ID NO:22).

Antibody preparation—For bacterial production of IL-20, an open readingframe coding for the mature form of IL-20 (residues Q21-F180 of SEQ IDNO:2) as predicted by SignalP (See, Nielsen, H., et al., Protein Eng.12:3-9 (1999)), was amplified by PCR and cloned as an Ndet-Asp7181restriction fragment (495-bp product) downstream of an inducible lacZpromoter. For efficient translation, the first 50 nucleotides of matureIL-20 were codon optimized for expression in E. coli. The primers usedwere: sense, 5′-GAC TCA TAT GCA GCC GCG TTC CCC GAA ATC CAA GCG TAA A-3;antisense, 5′-GAC TGG TAC CTT ATC AGA AGA TGC AGG TGC AGC-3′. Thereading frame and adjacent areas were sequence confirmed followingcloning. After transformation and expression in E. coli, IL-20 waspresent in the inclusion bodies. Inclusion bodies were solubilized with4M guanidine HCl and dialyzed against 50 mM sodium acetate buffer, pH 5,containing 0.1 M NaCl. Antisera were prepared by immunizing rabbits withIL-20 (Q21-F180). The sera were used for immunoblot analysis after1000-fold dilution.

Cell culture—In vitro cultures were grown in sterile disposablepolystyrene (Corning Glass Works, Corning, N.Y.) in a humidifiedatmosphere with 5% CO₂. 293, CE-10, NIH3T3, WRL-68, Colo587, PANG-1,HeLa S3, K562, Raji and SW480 cell lines were obtained from the AmericanType Culture Collection (ATCC™).

Transient transfections—Plasmid DNA was transfected into 293T cellsusing LipofectAMINE reagent (LIFE TECHNOLOGIES™, Rockville, Md.)according to the manufacturer's instructions.

Generation of stably transfected CHO clones—The complete open readingframe of human IL-20 was amplified by PCR. The primers used were: sense,5′-GAC TOG ATC CGC CAT CAT GGA CTG GCC TCA CAA CC-3 (SEQ ID NO:25);antisense, 5′-GAC TGG TAC CGG ATG GTC TCG GGC TGC TG-3′ (SEQ ED NO:26).Full-length IL-20 was cloned as a Bam II-Asp718I restriction fragmentinto a CMV-Enhancer/RSV-LTR promoter-based expression vector. The cloneswere sequence confirmed before transfection into CHO cells. IL-20positive CHO clones were selected by RT-PCR and amplified to 1micromolar methotrexate. Conditioned media (CHO-5 serum-free mediawithout methotrexate) from 7 CHO clones were analyzed for IL-20expression by SDS-PAGE followed by silver staining. Three CHO cloneswith the highest expression were selected for continued amplification inthe presence of 10 micromolar methotrexate.

Purification of IL-20—Four day conditioned media from IL-20 expressingclones was used for protein purification. The media was adjusted to 25mM HEPES buffer, pH 7.2 and applied to the strong-cation exchange resin(Poros HS-50) using a BioCad 60 (PEPerseptive). The HS-50 bound materialwas eluted using a step gradient of NaCl in 25 mM HEPES buffer, pH 7.2and fractions analyzed by SDS-PAGE. The 0.8M NaCl pool was applied toweak anion-exchanger (CM HyperD, BioSepra) and eluted with a NaClgradient. The IL-20 positive fractions were pooled, subjected tosize-exclusion chromatography on a Superdex 75 column (PHARMACIA™)equilibrated with PBS and pooled again. Protein concentration wasdetermined using the BCA procedure (Pierce Chem. Co). Endotoxin wasmeasured using the LAL assay (Cape Cod Assoc.).

Purification of epitope-tagged IL-20—For synthesis of N-terminal Flagfusion protein, the mature portion (nucleotides 105-584 of SEQ ID NO:34)coding region of IL-20 was amplified by PCR and cloned into pFLAG-CMV-Ivector (SIGMA™, Saint Louis, Mo.) as an EcoRI-BamHI restrictionfragment. The primers used were 5′-GCC CCG GAA TTC AAG GAG CCC CAA AAGCAA GAG G-3′ (SEQ ID NO:27) (sense) and 5′-GCC CGC GGA TCC TCA GAA GATGCA GGT GCA GCC-3′ (SEQ ID NO:28) (antisense). Conditioned media from293T cells transiently transfected with pFLAG-CMV-1:IL-20 was preparedand purified using anti-Flag affinity chromatography according to themanufacturer's instructions. Approximately 300 micrograms of purifiedprotein was recovered from 500 ml of culture supernatant.

IL-17R and IL-17RLP Purification—The extracellular portion of eachreceptor was fused to a human Fc domain (heavy chain constant region ofIgG1). The primers used for PCR amplification of the extracellulardomain coding region of huIL-17R were 5′-GAT CGC GGA TCC GCC ATC ATG GGGGCC GCA CGC AGC CCG CCG TCC G-3′ (SEQ ID NO:29) (sense) and 5′-GAT CGCGGA TCC CCG TCC GGA ATT GGT TCT GGA GTG TCT GGC ATT TCT G-3′ (SEQ IDNO:30) (antisense), and 5′-GAG CGC AGA TCT GCC ACC ATG TCG CTC GTG CTGCTA AGC CTG G-3′ (SEQ ID NO:31) (sense) and 5′-GGG GGG AGA TCT CCT CCCGGC TTG CTT TTG TTG TTA TC-3′ (SEQ ID NO:32) (antisense) for huIL17RLP,respectively. Clones with correct insert orientation were selected byPCR screening and resequenced before use. Conditioned media from 293Tcells transiently transfected with the IL17RLP (Met-(−19) throughGly-270 of SEQ ID NO:2)-Fc fusion or IL-17 receptor (Met-1 throughAsp-315)-Fc were prepared. The Fc-protein was purified using a Protein Acolumn (POROS), and approximately 150 micrograms of purified protein wasrecovered from 500 ml of culture supernatant.

Binding Analyses—IL-20 protein was dialyzed against 10 mM sodium acetatebuffer, pH-5 and a BIACORE™ flow cell was prepared for each receptor atdensities of 7900 and 9600 RU for IL-17R and IL17RLP, respectively.Various concentrations of purified IL-20 and IL-17 (R&D Systems,Minneapolis, Minn.) in 50 microliters hepes-buffered saline (HBS) bufferwere examined for receptor binding at a flow rate of 15 microliters perminute. After injection of sample the flow cell was equilibrated withHBS. Flow cells were regenerated using two 40 sec pulses of 10 mM HCl.

Flow Cytometric Evaluation of IL17RLP transfectants—For detection ofIL-20, cells (10⁶ in 100 microliters) were incubated with eitherpre-immunized rabbit serum (1:100) or IL-20 immunized rabbit serum(1:100). Cells were washed, then incubated with PE conjugated goatanti-rabbit F(ab)₂. Cells were washed, resuspended in 5 micrograms permilliliter propidium iodide solution and acquired on the FACScan (BectonDickinson Immunocytometry Systems, San Jose, Calif.). Alternatively,cells were first incubated 10 minutes at room temperature with 1microgram soluble IL-20, then the anti IL-20 serum was added asdescribed. Ability of soluble IL17RLP-Fc to block IL-20 binding toIL17RLP positive cells was also tested, using 1 microgram or 10micrograms IL17RLP-Fc added in solution with the soluble IL-20. Analysiswas performed using an electronic gate on propidium iodide negative livecells.

Peritoneal Exudate Cells—BALB/c mice (n=8 per group) were injectedintraperitoneally with 0.2 ml of rhIL-20 at indicated amounts plus 50micrograms of the human chemokine HCC-1 (See, Schulz-Knappe, P., et al.,Exp. Med. 183:295-299 (1996)) as a carrier. At 4 h after injection themice were sacrificed by CO₂ asphyxiation. The peritoneal cavity was thenexposed and the exudate collected by washing the cavity with 4 ml ofPBS. Cell counts performed in triplicate on each peritoneal exudatesample were quantitiated by complete blood chemistry (CBC) analyzer andhemocytometer. Cytocentrifuge (Shandon, Inc., Pittsburgh, Pa.) smears ofPEC from each mouse were stained with Wright's stain for differentialcounts. Total numbers of PMN accumulating in the peritoneal cavity werecalculated by multiplying total PEC by the percent PMN determined fromdifferential counts. Both percent and total values of PMN were expressedas the mean+SEM. Significance of difference was determined by an ANOVAt-test.

Reagents—Recombinant human IL-17 was from R&D Systems (Minneapolis,Minn.). Dextran sulfate sodium (DSS, 36,000-44,000 Mol. Wt) waspurchased from American International Chemistry (Natick, Mass.).LipofectAMINE reagent and geneticin (G418) were from LIFE TECHNOLOGIES™(Rockville, Md.). Indomethacin and methotrexate were obtained fromSIGMA™ Chemical Company (St. Louis, Mo.).

Animals—Female Swiss Webster mice (20-25 g) and female Lewis rats(160-180 g) were obtained from Charles River Laboratories (Raleigh,N.C.) and kept under standard conditions for one week before being usedin experiments. The animal protocols used in this study were reviewedand approved by the Human Genome Sciences, Inc, Institutional AnimalCare and Use Committee.

Tissue Collection—Tissues from several models of inflammation weretested for expression of the IL17RLP. Models included murine colitis,rat jejunitis, mouse graft versus host disease and Listeria inducedbacteremia in mice.

In the dextran sulfate sodium (DSS) induced murine colitis model, femaleSwiss Webster mice were given a four percent solution of DSS ad libitumfor seven days. Animals were euthanized on day seven, and the distalthird of the colon flushed with saline and snap frozen with liquidnitrogen in preparation for RNA extraction.

In indomethacin induced rat jejunitis, female Lewis rats were injectedsubcutaneously on day 0 and 1 with indomethacin. Indomethacin wasprepared by solubilizing in absolute ethanol, sonication for 30 secondsand then diluted 1:4 vol/vol with five percent sodium bicarbonate tocreate a stock solution of 10 mg/ml. The stock solution was dilutedfurther with five percent sodium bicarbonate, and rats were injectedsubcutaneously (sc) with a final dose of 8 mg/kg in a volume of 0.2 ml.On day 4, three days after the final indomethacin injection, rats wereeuthanized, and 10 cm of the small intestine removed, starting 20 cm upfrom the cecum. The intestinal tissue was flushed with saline and snapfrozen with liquid nitrogen prior to RNA analysis.

Primary structure of IL-20—An EST coding for a putative signal peptidewas initially discovered in a human thymus cDNA library. Threeadditional clones were subsequently identified in libraries from thymustumor and from 9 and 12 week old human embryo tissue. All four cloneswere fully sequenced and found to be identical over the entire openreading frame. They are predicted to code for a protein of 184 aminoacids with an N-terminal leader sequence of 20 amino acids (SEQ IDNO:35). The predicted molecular mass for this protein is 20.4 kDa, withan estimated isoelectric point of 924. There is one potential N-linkedglycosylation site and eight cysteine residues. The short 3′untranslated region contains a single near-consensus polyadenylationsite and is devoid of the characteristic AU-repeats found in severalother cytokines, growth factors and protooncogenes (See, Shaw, G., etal., Cell 46:659-667 (1986)).

A comparison of both nucleotide and amino acid sequences with theGenBank or EMBL databases revealed significant homology of thetranslation product with the amino acid sequence of the recentlydescribed T cell-derived cytokine, IL-17. At the amino acid level, humanIL-20 shared 21.3%, 19% and 20.7% identity with human, mouse and ratIL-17, respectively, and 21.9% identity with the product of the 13th ORFof Herpesvirus saimiri (HVS13). The degree of conservation is higher inthe C-terminal portion of the protein, and six of the eight cysteinespresent in IL-20 are conserved and identically spaced between IL-20 andIL-17 (See, Yao, Z., et al. and Fossiez, F., et al., supra). A putativemurine ortholog of IL-20 was identified in a mouse EST database andfound to be 87.8% similar to the human IL-20 and 21.3%, 19.6%, 22% and21.9% similar to the human, mouse, rat and viral IL-17 sequences.

The map position of the human IL-20 gene was determined by somatic cellhybrid and radiation hybrid mapping. Amplification of the standard G3radiation hybrid panel using gene specific oligonucleotide primersshowed linkage to the SHGC-33930, SHGC-4655 and SHGC-11215 markers onchromosome 6 at distances of 13, 14, and 18 centiRad, with LOD scores of10. 13, 9.25, and 8.94, respectively, corresponding to a cytogeneticlocation at 6p21.2.

Cellular and tissue distribution of the hIL-20 mRNA—By Northern blotanalysis of human tissues, a very strong signal at ca. 1.0 kb was seenin spinal cord, testis and small intestines, and less pronounced inprostate, colon mucosal lining, ovary and in the K-562 chronicmyelogenous leukemia cell line. Furthermore, a weak transcript ofsimilar length was routinely observed in trachea, uterus, adrenal gland,substantia nigra and fetal kidney. Even though IL-20 cDNA was initiallyisolated from thymus, the signal observed on all blots with spleen orthymus poly-A RNA was either feint or not visible. The tissuedistribution of murine IL-20 was also determined. A ca. 1.0 kb band wasobserved on poly-A mouse RNA blots probed with a murine readingframe-specific cDNA probe. The signal was strongest in brain, heart andtestis and weaker in lung, liver and skeletal muscle.

Molecular characterization of an IL-20 receptor—In order to identifytarget cell types that respond to IL-20, we searched for candidatereceptors. Since IL-20 is distantly related to IL17, we screened the ESTdatabases for novel homologs of the recently described murine and humanIL-17 receptor amino acid sequences (See, Yao, Z., et al., Immunity3:811-821 (1995); Yao, Z., et al., Cytokine 9:794-800 (1997)). A cDNAclone containing an open reading frame predicted to code for a type Itransmembrane protein was identified in a library from human adult lungtissue. Overlapping clones were subsequently discovered in librariesfrom various other tissues, predominantly eosinophils, brain, pancreas,kidney, thyroid and osteoclastomas. A large open reading frame ispredicted to encode a receptor of 426 amino acids. Computer-assistedanalysis suggests that this protein has an N-terminal signal peptidewith a cleavage site after Pro-(−3) of SEQ ID NO:2. The signal peptideis followed by a 273 amino acid residue extracellular domain (Arg-(−2)through Gly-270 of SEQ ID NO:2), a 22 amino acid residue transmembranestretch (Trp-271 through Leu-292 of SEQ ID NO:2), and a 115 amino acidresidue cytoplasmic tail (Met-293 through Leu-407 of SEQ ID NO:2). Thereare six potential N-linked glycosylation sites in the extracellulardomain, at positions Asn-48; Asn-84; Asn-137; Asn-164; Asn-178; andAsn-264 of SEQ ID NO:2. The predicted molecular mass for this protein is47.9 kDa, with an isoelectric point of 8.16. Overall, the IL17RLPprotein sequence is 19.2% and 18.2% identical to the human and murineIL-17R sequences, respectively. There is no WS×WS motif in theextracellular domain (See, Baumgartner, J. W., et al., J. Biol. Chem.269:29094-29101 (1994)). The cytoplasmic portion of this new receptor ismuch shorter than the unusually long tail described for IL-17R (See,Yao, Z., et al., Cytokine 9:794-800 (1997)). Furthermore, a segment(TPPPLR-PRKVW) (SEQ ID NO:33) located proximal to the IL-17Rtransmembrane domain, which is highly conserved among cytokine receptors(See, Baumgartner, J. W., et al., J. Biol. Chem. 269:29094-29101(1994)), is absent.

By Northern blot analysis of human tissues using an open reading framespecific hybridization probe, two specific transcripts of ca 3.5 kb and1.4 kb can be detected in several endocrine tissues, most pronounced infetal and adult liver, kidney, pancreas, testis, colon and smallintestines but are absent in peripheral blood leucocytes and lymphoidorgans. Only a few of a large series of transformed human cell linesgrown in culture expressed IL17RLP transcript detectable by Northern andreal-time PCR analysis. These were predominantly derived from organsfound to be positive for IL17RLP message above and included the WRL-68human embryonic liver, Colo587 pancreas adenocarcinoma-mesothelioma,PANC-1 pancreatic epitheloid carcinoma, HeLa S3 cervical carcinoma, K562leukemia, Raji Burkitts lymphoma and SW480 colorectal adenocarcinomalines.

The map location of IL17RLP was determined at 3p21.1 by radiation hybridmapping, with a LOD score of 12. It is noted that several chemokinereceptors and trypsin inhibitors have been mapped in the 3p21.1, 3p21.2and 3p21 regions.

Tissue distribution of rodent IL17RLP—Many cytokine receptors are notexpressed constitutively, but their transcription and surface expressionis dependent on specific activation mechanisms. To gain insight intopossible roles of this novel cytokine receptor pair in diseaseprocesses, a partial cDNA clone for the putative murine IL17RLP orthologwas identified and hybridized with total RNA prepared from a series ofrodent disease model organs. Because of the proinflammatory roles ofIL-17, RNAs from several inflammatory models were used. These includedkidney and liver RNAs from a murine model of graftversus-host disease,liver following Listeria-infection, as well as colon and intestinaltissues from DSS-induced colitis and from Indomethacin-inducedintestinal inflammation in rats. Among the models tested, IL17RLPmessage was found to be significantly upregulated only in the intestinesafter Indomethacin treatment. However, the upregulation was drastic,from weak or undetectable in most untreated samples to a readilydetectable or intense signal in total RNAs from several differenttreated animals. As seen above with the human probe and human tissues,two transcripts of 3.4 kb and 1.3 kb were observed.

Expression of recombinant IL-20 protein—Human IL-20 was cloned asdescribed in experimental procedures and expressed in CHO cells underthe control of an RSV-CMV hybrid promoter. Comparison of the proteinpattern by SDS-PAGE analysis of conditioned media from IL-20 clonesversus the control media revealed that several clones expressed a novelprotein of ˜20 kDa not present in control media transfected withexpression vector plasmid only. One clone was chosen for scale-up andconditioned media were obtained after 4 days. Immunoblot analysis ofconditioned media using a polyclonal antibody revealed the presence ofseveral species of IL-20, which suggested the presence of proteolyticprocessing and/or differential glycosylation of the protein. IL-20 waspurified to apparent homogeneity as described in ExperimentalProcedures. PAGE analysis of purified IL-20 under non-reducingconditions showed that, unlike IL-17, IL-20 migrates as a monomer andthus is not a disulfide-linked dimer under these conditions. However,when eluted from a Superdex 75 size exclusion column, IL-20 behaves as adimer. Thus, native IL-20 appears to be a non-disulfide linked dimer.

The major bands were subjected to N-terminal sequence analysis. The23/22 kDa species had four closely spaced N-termini starting at Arg-23,Ser-27, Arg-29 and Lys-30 of SEQ ID NO:35 (in roughly equal proportion),whereas the 18 kDa band had two N-termini starting at residues Leu-49and Ser-51 of SEQ ID NO:35. The presence of truncated forms of theprotein is suggestive of posttranslational proteolytic processing. Thisappears to be due to the action of a proprotein convertase-like activityas three of the N-terminal residues Arg-29, Ser-30 and Met-52 of SEQ IDNO:35 are preceded by basic residues. However, Ser-51 of SEQ ID NO:35 ispreceded by Val and may not be processed by the same enzyme. Whenexpressed in baculovirus, only one species was detected. The N-terminusof baculovirus expressed IL-20 was Arg-23 of SEQ ID NO:35, which is tworesidues downstream of the cleavage site predicted by SignalP (See,Nielsen, et al., supra.), Gln-21 of SEQ ID NO:35. Thus, IL-20 isolatedfrom CHO conditioned media appears to occur in several forms due toposttranslational proteolysis. The effects of processing on biologicalactivity are not yet known.

Binding experiments—Specific interaction of the extracellular domain ofthe novel receptor with soluble IL-20 purified as described above wasdemonstrated independently by three different methods. The predictedextracellular domains of human IL-17 receptor and of IL-20 receptor wereexpressed as chimeric proteins, fused to the heavy chain constant regionof IgG1. When used as immobilized component in the BIACORE™ surfaceplasmon resonance analysis system, purified soluble IL-20 bound toIL17RLP, in a concentration-dependent manner. Very poor interaction ofthis receptor was observed with soluble recombinant human IL-17. Incontrast, IL-17 bound well to IL-17 receptor under the same experimentalconditions.

293T cells transiently transfected with human IL17RLP expression plasmidwere used to measure cell surface binding of IL-20 by flow cytometry asdetected by an IL-20 antibody. Significant binding of IL-20 was observedin the IL17RLP transfectants but was undetectable in untransfectedcells. IL-20 antibody alone did not bind to untransfected or transfectedcells. Furthermore, cell surface binding was inhibited by the additionof soluble IL17RLP-Fc fusion protein. This inhibition of binding wasdose dependent, as the mean fluorescence peak was shifted back by 15%and 90% by the addition of 1 microgram and 10 micrograms of receptorprotein, respectively.

Specific cell surface binding of epitope-tagged IL-20 was alsodemonstrated. The SW480 colorectal adenocarcinoma cell line, shown aboveto express IL17RLP transcript, was used in this experiment. Binding ofN-terminal Flag-IL-20 fusion protein to these untransfected cells wasdetectable as a quantitative shift after staining with Flag- orIL20-specific antibody, in contrast to the only partial shift observedwith the transfected cell population above.

Finally, binding of huIL-20 to huIL17RLP was confirmed byco-immunoprecipitation. Purified IL17RLP-Fc fusion protein was incubatedwith soluble CHO-derived recombinant human IL-20. Binding of IL-20 toIL17RLP was demonstrated by detection of IL-20 in the protein A agarosecoprecipitate by Western immunostaining.

Neutrophil migration elicited by IL-20 in vivo—Treatment with IL-17 hasbeen shown to activate the transcription factor NF-kappaB and to induce,cytokine secretion in fibroblasts (See, Yao, et al., J. Immunol.155:5483-5486 (1995); Fossiez, et al., J. Exp. Med. 183:2593-2603(1996)). In our hands, treatment with CHO-expressed and purified IL-20did not activate NF-kappaB in NIH3T3 cells. Furthermore, no reproducibleinduction of cytokine message or protein (e.g., IL-6, IL-8, TNF-alpha,IFN-gamma, IL-3, G-CSF) was observed in HeLa, CHO or 293T cells aftertreatment with rhIL-20 in vitro.

To examine its possible physiological roles in vivo, recombinant humanIL-20 was injected into BALB/c mice. As the abundance of IL-20transcripts in RNA from colon mucosal lining and small intestines maysuggest functions of the cytokine on the gastrointestinal tract walls,intra-peritoneal (i.p.) injection was chosen as the route ofadministration. The results demonstrate that i.p. injection of rhIL-20consistently caused a dose-dependent influx of PMN into the peritonealcavity within 4 hours. This influx of PMN was not a result ofnon-specific vascular leakage because very few RBC were observed in mostcytopreparations. Red blood cells or clotting visible in some animalswas attributed to vasculoepithelial injury during injection, and thesepreparations were excluded from analysis. Another cytokine, rhHCC-1(Schulz-Knappe, P., et al., Exp. Med. 183:295-299 (1996)), showed noeffect on PMN infiltration, even over a wide range of proteinconcentrations, and therefore was chosen to serve as protein carrier forthe low dose study of IL-20. Peritoneal PMN infiltration was stillmarked in response to 100 ng of L-20 per mouse but became statisticallyinsignificant at 10 ng. The results are not attributable to LPScontaminants since (a) the amount of LPS in rhIL-20 is ten fold lowerthan that of rhHCC-1, and (b) heating of rhIL-20 at 80° C. for 45 mincompletely abrogated its ability to cause PMN influx.

Several observations in this report suggest the physiological role ofIL-20 to be distinct from IL-17 or other previously described secretedfactors. First, while IL-17 is found to be expressed almost exclusivelyby CD4+ and DN activated T cells (See, Yao, et al., J. Immunol.155:5483-5486 (1995)), IL-20 is highly transcribed in human and murinespinal cord, and low levels of expression can be found in many otherorgans. Second, the AU-rich repeats indicative of transient expressionfound in IL-17 and other cytokines are absent from the 3′ untranslatedrealon of the IL-20 transcript (See, Shaw, G., et al., Cell 46:659-667(1986)). IL-20 may thus be the translation product of a more stablemessage that in fact could give rise to a constitutive serum presence ofthe protein. Third, while a specific cell surface receptor for IL-17 isdescribed to be expressed in virtually all cell types (See, Yao, Z., etal., Immunity 3:811-821 (1995)), the receptor for IL-20 discovered hereshows a highly specific message expression pattern, largely restrictedto kidney, liver, pancreas and intestines. Furthermore, the drasticallyshorter cytoplasmic tail of IL17RLP as compared to human and mouseIL-17R indicates that there may be principal differences in thecorresponding downstream signalling processes.

Recombinant human IL-20 protein did not exert any detectable chemotacticactivity upon peripheral blood neutrophils or eosinophils from severalhuman donors. Moreover, IL17RLP message was undetectable in humanneutrophils by either Northern or real-time PCR analysis, andneutrophils failed to bind epitope-tagged recombinant IL-20 by FACSanalysis. Therefore, the dose-dependent neutrophil influx observed afteri.p. injection is unlikely due to a direct activity on neutrophils.Rather, IL-20 binding to cell surface receptors on stromal or otherconnective tissue elements may trigger expression and secretion ofchemoattractive factors from these cell types, leading to a guided localaccumulation of poly morphonuclear leucocyte populations. Accordingly,our inability to observe transcription factor activation or mRNA andprotein expression of several known chemokines in transformed cell linesin culture is most likely due to a requirement for a specific activationprocess to render cells responsive to IL-20. In addition, IL-20 couldallow or enhance migration of polymorphonuclear cells into thegastrointestinal tract, or other epithelial structures by acting not onthese invading cells directly, but via some effects on the localmicrovasculature of these tissues. However, even though recombinantexpression of IL17RLP cDNA alone is sufficient to yield specific cellsurface binding sites, it cannot be ruled out that IL-20 acts on thesecells by additional receptors or receptor components.

Because of the similarity of IL-20 to the pro-inflammatory cytokine,IL-17, and its association with neutrophil chemotaxis, IL17RLP messagedistribution studies were conducted in target tissue from various modelsof inflammation. Among those models were DSS induced colitis andindomethacin induced jejunitis, both models of inflammatory boweldisease (IBD). Although Northern blot analysis showed no IL17RLP in thecolons of DSS treated mice, IL17RLP was dramatically upregulated on day4 in the mid bowel of rats receiving consecutive indomethacin injectionson day 0 and 1. Indomethacin induced jejunitis is characterized bytransmural lesions and an influx of neutrophils. Though there is littleevidence for an immunologically driven mechanism of action,indomethacininduced IBD bears some resemblance to Crohn's disease, it'sclinical counterpart, in that it: a) induces transmural lesions; b)causes non-bloody diarrhea; c) has a genetic component; d) is dependenton the presence of bacteria; e) causes granuloma formation and 1) isaccompanied by inflammation (See, Kim, H, et al., Scand J.Gastroenterol. 27:529-537 (1992); Elson, C. O., et al., Gatroenterol.109:1344-1367 (1995)).

Thus, as described herein, IL-20 and IL17RLP is a novel cytokineligand-receptor system that may be involved in specific localinflammatory processes and in the indirect recruitment of neutrophils totissue repair and immune reactions at specific target organs.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

Further, the Sequence Listing submitted herewith (in both paper andcomputer readable forms), and the Sequence Listings submitted with U.S.application Ser. No. 09/796,844, filed Mar. 2, 2001, U.S. ProvisionalApplication Ser. No. 60/187,015, filed on Mar. 6, 2000, PCT ApplicationSerial No. US00/05759, filed on Mar. 6, 2000; PCT Application Serial No.US99/21048, filed on Sep. 15, 1999; U.S. application Ser. No.09/268,311, filed on Mar. 16, 1999; PCT Application Serial No.US98/19121, filed on Sep. 16, 1998, U.S. application Ser. No.09/154,219, filed on Sep. 16, 1998, and U.S. Application Ser. No.60/059,133, filed on Sep. 17, 1997, in both computer and paper forms arehereby incorporated by reference in their entireties.

What is claimed is:
 1. A kit comprising (i) an isolated antibody orfragment thereof that binds specifically to an Interleukin 17Receptor-Like Protein, wherein the Interleukin 17 Receptor-Like Proteinconsists of an amino acid sequence selected from the group consistingof: (a) the amino acid sequence from position −19 to position +407 ofSEQ ID NO:2; (b) the amino acid sequence from position −18 to position+407 of SEQ ID NO:2; (c) the amino acid sequence from position +1 toposition +407 of SEQ ID NO:2; (d) the amino acid sequence from position+1 to position +271 of SEQ ID NO:2; (e) the amino acid sequence fromposition −5 to position +271 of SEQ ID NO:2; (f) the amino acid sequenceof the full length protein encoded by the cDNA in ATCC Deposit No.209198; (g) the amino acid sequence of the full length protein,excluding the N-terminal methionine residue, encoded by the cDNA in ATCCDeposit No. 209198; and (h) the amino acid sequence of the extracellulardomain of the protein encoded by the cDNA in ATCC Deposit No. 209198;and (ii) an isolated polypeptide comprising a portion of SEQ ID NO:2 ora portion of the protein encoded by the cDNA in ATCC Deposit No. 209198which binds specifically with said isolated antibody or fragmentthereof.
 2. The kit of claim 1, wherein said kit further comprises acontrol antibody which does not react with said polypeptide.
 3. The kitof claim 1, wherein said antibody or fragment thereof is conjugated to adetectable substance.
 4. The kit of claim 3, wherein said detectablesubstance is selected from the group consisting of a fluorescentcompound, an enzymatic substrate, a radioactive compound and aluminescent compound.
 5. The kit of claim 1, wherein said polypeptide isattached to a solid support.
 6. The kit of claim 1, wherein saidantibody or fragment thereof is attached to a solid support.
 7. The kitof claim 1, wherein said antibody or fragment thereof is a monoclonalantibody.
 8. The kit of claim 1, wherein the antibody or fragmentthereof is a polyclonal antibody.
 9. The kit of claim 1, wherein theantibody or fragment thereof is a human antibody.
 10. The kit of claim1, wherein the antibody or fragment thereof is a chimeric antibody, ahumanized antibody, or a single chain antibody.
 11. The kit of claim 1,wherein the fragment is a Fab fragment.